Note: Descriptions are shown in the official language in which they were submitted.
~89~7
_ 1 _
DE SCRIPTION
~IETHOD FOR DE'rECTING, IDENTIFYING, A~D
QUA~TITATING ORGANISMS AND VIRUSES
TEC~ICAL FIEI,D
S T~ ~v~tion relates eo a me~ d ~ru f~r deteoting,
is~entifying, and ~antitatin8 orgs~ in biolo~Lcal ~1 other
sa~les. lta~s, lt relate~ to a T~thod for ~pec~f~ lly and
iti~rely detecting and quantitating ~ org~; c~ ~e
r~os~l R~, (hereinafte~ R-R~), transfer RNA (h~eir~:~t~ t-R~A)
cate~ie~ or t~Lc 3ro~p3 of ~h organis~ and pr~vi~ly
ur~ org~ c~ntaining R-g~ or t-~. The method i~ capable
of da~ct~ preqer~:e of ev~ c~e or~, caitainin8 R-RNA
11~ i~tia~ al30 ~lve-~ a m~t:hod for uuing spec~fically
prcx~ced nucle~c asid3 ca~?l~ntary to specifi~ seq~su::e~ or
~ti4r~ of di~ferent s~e~ of the ~ clas3 ~NA, cr
~; hr~A, or sr~ or the cla~ of ~NA se~ce3 ~herelslaftec prec~so~
specific seq~:es or b~dgEaA) ~h æe pre~ent ally in the pre-
cursor ~A, R~ , t-R~A, hr~ oq: ~A ~l~cule~, ant noe in
re ~IA, R-}~A, t-R~,.~ c1r sr~A ~Dlecules r eo det~t,
id~ti~y, ~d ~i~te ~ organi~, groups of c~gani~s,
thereof can be m~re cle~ly u~c~ood and a~preciated wh~
ca~it~ed in 1~ of ~e repre~en~tit7e ba~egrould inf=~
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BACXG~O~ ART
Each of t~ cells of all life for~, except vi~ , cantain
sep~ra~e ingl@ ~tr~ld E~ rrDlecules, namel~, a læg~e ~lecule, a
5 ~ un s~z~d ~lecule, and a s~all ~lealle. The t~ larger R-RNA
Ribos~l RNA i~ a d~rect ~ pro~ct znd i~ c~ or by
the R~ . ~ ~A ~ce ~s used ~ a t~late ~
sgn~he~ize R-R~A mD~ e3o A sepæate ge~ or each of
10 ~he ribo3a~1 RNA s~i~. Mhlt~ple R R~ gena~ eYdst in ~st
cb~i~l R~ . Pl~nt8 ar~ c~rtai~l o~r form~ caAta~
~clesr, ~itx~sl d chlaropla~t ~-R~ gene~. For slmp~lc~
o di~als ion h~re~na~, the t~ee ~ep~ate R~aA gene~ will be
15 refsrred to a~ t~e R-R~A gQ~e.
A~ut 85-90 p~c of ~ tot~ E~ i~a a typi~l eell ~ R~
A bacteria ~h ~ ~. ~li c~tain~ 104 ril:osa~ per cell
a ~lL~ liver oell contaisu al~ 5 x 10~ r~o~. S~e
~0 each ri~osom2 coatai~ or~ d esch R-R~ g~ he bac~ l cell
ar~ ~ 104 ar~l ~ x 10~, ~especti~7ely~ of each
R-~A ~it.
25 G~ 1~ ~ti~8 o~ a p~t~ nu~ cld ~e, e~
~d~ae~ æe f~, far ~le, in
~c e~e~R of ceLl~ ~8l ~retic analy~i~ of lii~e
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Probably the best ch~acterized and ~ct stu~ied gene ant
gene product are *~e R~ A ~e and R-R~3A, and the priar art Includes
use of ~ybridizacion of R~ d ribosanal geres ~n gene~ic dysis
ar~ e~lution and ra~or~ classifis:atic~ of ar~ a~d r~bosa~
5 g~ne se~:e3~ Genetic dycis in~ or ~ple, the deter-
m~atic~ of ~e ~ers of ribasarlal R~ gena~ in væious ar~an:~;
the dete~nat~an of the similarity bet~æen the ~ti~le ribo=al
RNA genes which ~e present in cell~ ~tian of the~ rate
and e~ctent of synthe~eis Of R-RNA in cell~l and ~ faceors whichO control them, E~lution ~ul eaxon~ ~udies i~}volve ca~æ~
R-Rl~ 8en~ base sequ~e ~ related and widely different
~gan~. ,
It i~ Icnawn ~:hat the ribo~al R~ ~ene ba~e seq~ce ~ a
least p~ Llly simil~ in w~dely d~ ent organ~ms, ans!l tha~ the
15 ~ of coli bacte~al r~bow0al R~ gen~8 ~dize~ well with
R-~NQ from plant~ and a w~de variet~y o~ o*~e:r bæteri~l
s~c~ ti~n of the E:. col~ geae ~lch h~e9 tO
t~ other sp~cies varieq wi*~ ~he degreQ of relat~ess of the
argE~. Vir~ually 211 of the R~ ~e ~ce ~rldizes
20 to R-~&~. i~ clo~ly related bsc~rial speci@~, ~hile les~
~hridize~ to ~-R~ ~r~ distanely related bæterlal spec~es, and
ev~ les8 wl~
As wi~h R-RNAs, .t-E~ æe pr@~ is all liv~ cells, a~
~æll as in ~ v~ase~. t-~ genes ~e p~esen in c~os~al
25 and pl~ ~8 o~ prok~y~te3 and in ~æ ~ o e~caryotic cells,
includ~ ~he ~ of the T~c~ ~it~ia and chloroplasts.
~f~rent t~ ar cne t-R~A species of~n exist in a single
cell. S-R~ gene~ of alitoc~n~ria, ~n~leic ~nd chloropla~sts æe
30 ~ eo th0 viru~.
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t-RNA molecules are direct gene.products and are
aynthe3ized in the .c~ using ehe t-RNA gene a~ a
templaee. The t-RNA i~ of~cen synthesized a~ part of a
larger ~IA. ~olecula, and the t-RNA portion i~ ~hen removed
5 fro~ thi~ precur~or molecule. After synthesls a fractlon
of the bases of the ~-~IA molecule are chemically modified
by t~le cell. A typlcal t-RNA molecule contains from 75-85
ba~e ~ .
Nu~erous ~-RNA molecul~s are preRent in all cell~
10 of all life for~, and uQually abou~ 10 percent of a
cell ' g ~cotal RNA i9 compo~ed of t-RNA, a typical
bacterial cell contain~ng about 1.5 x 105 c-RNA molecule~
of all types. If ~ach differen~ kind of t-RNA i3 equally
represented in a bacter~al cell then 2500 of each d~fferent
15 t-RNA Dlolecule i9 pre~ent in each cell. A t~ical ~ammalian
liver cell contains about 108 t-RNA molecule~ or an average
of about 106 copies per cell of each different t-3~NA type.
During prs~eirl ~ynthesi~ individual a~no a id~ are
ali~ned in the proper order by variou~ ~pecif ic t-RNA~,
20 each amino acid being o~dered by a differ~nt t-RNA specie
So~e amino acids are ordered by ~or~ than one t-RNA t~rpe.
: ~ : There are cer~ai~ viru~e~ which contain t-RNA gene~
~: ~ in ~heir genomQ~, ~hese ~ene producing ~r~ru~ -~pecific
t~RNA wher~ the viru~ g~no~e ls ~ctive in a cell. ThesQ
25 t-RNA~ can al o be prc~ent ~n multipIe copieq in each
~nfected cell.
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789~37
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A~ with R-RNA gene~ and R-RNA9 the prlor art
discloses u~ of hxbridlzatlon of t-RNA and t-RNA
genes in gen~tic analy~i~ and evolution and taxonomic
cla~sification o~ organi~s and t-RNA gene ~equence~.
5 Genetic analysis include~, for example, the deterr~
nation of the nu~ber~ of t EINA gene~ in variou~ organ-
ism~; the determination of che ~i~nilarlty between the
mult~ple t-RNA gene~ which are pre.~ent in cells; ~
dQter~ination of the rate and exten~ of syntheqi~ of .
10 t-RNA in cells and the factors whlch control theDa.
Evolution and ~axonomic -~tudie~ involv~ comparing the
t-RNA gene base ~equence from rela~ed and witely
different organi~
And a~ with R-R~ ~ene base ~equences, it ~ ~ ~nown tha~:
15 an individual t-RNA gene bsse sequence is at least
partlally si~ilar in dlfferent organi3m~. Total e-RNA
~how~ this a~ type of relationship and bulk t-RNA from
one ~peci~s will hybrld~ze ~ignlficarltl~ with t-RNA gene3
of a di~tantlsr r~lated o~ga~is~. Ral: D~itoehorldrial
20 leucyl-t R~ hy~r~d~z~t signiflcantly with T~itochontria
`, DNA of chickell a~d yea~ tBiocheD~ ry (1975) 14, tlO.
p. 2037). t-RN~ geT~e~ ve al~o been hown to be hlghly
corl~rved amon~3 ~h~ ~e:~ber~ of th~ baeterial fa~nily
En~erol~-cce ~e. Bull~ t-R~A genes fro~ E. col~
~5 hybr~dlze w~ll wi'ch t-RNA i~olatet ro~ 3pee~es repre- ~
sen~ 3 different gene~ (J. Bacceriology (1977) 129, ~3,
p. 1435-1439). Th~ fract~on of th~ E. coll t~RNA/gene~
~h~ch hybridizes to thes~ o her species varie~ w~th the
deg2ee of relate~;e~ of the orESan~ ~8 . A large fract~on
30 of ~che E .. col:L t~R~ gene seques~ce hstb~ dize~ eo ~-RNA
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from a closely related ~pecles while much le~s
hybridizet to R-RNA fro~a distantly related ~pecies.
The extent of conservation of the t-RNA gene
~equences during evolution is not a~ great as that
for the R-RNA gene sequence3. Nonetheles~ the t-RNA
gene sequenee~ are much ~ore highly conser~ed than the
vast bul~ of the DNA ~equences pres~nt ~n cell~.
The sen~itivity and ease of detection of mem~ers
of 3pecific groups of organlsms ~y utillz~ng probe
specif ic for th~ R-RNA or ~-RNA of that g;roup o~ organ-
isms i~ greatly enhanc~d b~ the larg~ nu~ber of both
R-RNA and t R~aA snolecules which ar~ present ~n each cell.
In addleion the hybr~ dization tes~ 1~ made ~ignifiean~ly
easier ~ince RNA molecules pre~ent ~n oell~ ~re ~ingle
s~randed. Thu~ a denaturation 8tep, such a~ must be
used for a hybridization te~t which detect~ any fraction
of cell DNA, is not neces~ary wh~n the tar8et molecule
i~ RNA. Probe~ specific foE other clas3es of oell nueleic
acld~, be31d~ R-RN~ or t-RNA, D~y be u3ed to specif~ eally
detect 9 ident$fy nd quanti~ate ~pccific group~ of
organlsTIl~ or cells by nucleic scid hybr~dization. Thus,
- other classe~ o~ p~okaryotic cells include
messenge2~ R~A (hereinafter ~), and RNA ~equence~
- ~ which are part of a var~ ~ty of precursor molecule~ .
2~ For exa~ple R-RNA i~ ~ynthe~izet in the bacteria E. coli
a a precur~or molecule about 6000 base~ long. Thi~
pr~cursor Ellolecule 1~ ~hen proce~3~d to yleld the R-RNA
subunits (totali~g about 4500 baqe~) which are lncorpor~
atet into ribo~o~ ~nd the exera R~ ~equence~ ( 1500
~ ~ 30 bas~s in ~cot~l~ which are discardedO t-RN~ ~aolecul2~
::~ and ribo~omal 5S RNA are also synthe~ized and proces~ed
uch ~ manrlerD
987
In prokar~otic cell-q infected by viru3e~ there
i3 al90 virus pecific mRNA pre ent. The DlRNAs of
certain prokaryotic v~ruses are ~lqo synthe~izet as
a precursor molecule which contains eXCesQ RMA ~equence~
5 which are er~ d away and dlscarded.
Many of the prokaryotlc mRllA3 and v~ nRNA~ are
present up to ~everal hunc~ed . time9 per cell ~hile
thousand.~ of the excess RNA ~eque~ce3 preQent in~-RNA
or t-RNA precursor mule~ules can be present in each cell.
Eukar~otic cells al80 contain preeurYor mRNA, a~
well as precur~or R-RNA and t-RNA, molecule~ wh~ch
are lar~er than the final R~ or t-R~lA. ~olecule~.
Irl contra~t to prokaryote~, many newly ~ynthe~ized
eukaryotic mRNA molecule~ are ~uch lar~ser than the
15 final mRNA ~olecule and contain exe@ss RNA -~equ~nce~
which are tr~ned away and ti carded. Another claqs
~ ~ o~ RNA. present ln eukaryotic cell~ i~ heterogeneou~
nuclear RNA (hereinafter known a~ hn-~), which i8
a di~erse cla~ oiE RNA which contain~ mRNA pr~cur or
20 lecule~ (which lea~e ths nucle~ ~For the cytopla~
where proteirl ~yrlthe~is occ~r~) and a larE~e amount of
RNA which nev~r leaYe~ the nucl~u3. Thi~ fraction also
corl~ain3 a 8mall fract~on of double ~rand RNA. Eukar-
yotic nuclel al~o contain ~all RNA molecule~ called
;~ ~5 Yalall nuclear RNA (hareinafter nRNA), varying in
length froffl 100-200 ba~e~.
The al:~undance " or rlu~ber o~ copies p~r cell, of
dif~erent 21~RNA molecules var1e~ eatly. Thi~ varies
:; froln a cQmpleac cla~3 o~ ~RNA molecules wh~ch are pre~ent
30 only 1-2 ~ per cell, ~o the mod~rately abundant
c1~3~ of RNA mol~cllles whlch ar~ pr~sen~c s~ver~l hundred
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times per cell, to the superabundant cla~3 o RNA
~olecule3 whlch may be present 104 or more times per
cell. Many of t~e RNA sequence~ present in hnRNA
are al90 very ab~ndan~ in each cell~ The RNA sequences
present in the precur30r RNA molecules for R-RNA, t-RNAs
and ma~y ~RNAs are also very abundan~ in each cell.
Individual snRNA sequence~ are extremely ab~ndant ant
say be present from 104 tO 106 time3 per cell.
Eukaryotic cells are al~o infected by ~iruse~
which produce ~rus sp~c~fic mRNA and in may cases
virus specific precux~or mRNA moleculeY which contain
RNA sequences not present in th~ ~ature mRNA ~olecule.
~he indlvldual viru~ speciflc ~RNA and precur~or RNA
molecule~ vary in abunda~ce from complex ~1-2 copies
: 15 per cell) to ~uperabundant (around 104 copie~ per cell).
My invention al80 r~late~ therefore, o a method
for 3pecifically an~ sen~it~ely detec~ing, identifying
and quantitat~ng or~a~ism~ ~ as well a8 ~ vlru~e~ ~ presen~
in cell~. Mor~ partic~larly, the method 1~ u3eful for
en~itively deteceing, identifying and quant~tating
any me~ber.o~.dif~erent .~lze~ categor~e~ of organi~
~; eukaryotic cell~, viru~, and ~n ~o~ ea~e prev~ously
unkn~wn organi3m~ containing ~RNA, hnRNA, snRNA or excesq
RNA ~olecules present in R-RNA, t~RNA, mRN~, or hnRNA
molecules.
Thi~ invention therefore ha~ broad application to
an~ area in which it i~ important ~o det~r~ine; the
pre~ence or ab~ence o ll~ing organi~m~, or ~iruses
pre~ene in cell~; ~he q~ate of ge~etic expre~ion of,
an orga~l~, cell, virus pre~nt ~n ~ cell, or groups of
~ .o~ pr~ol~o~lc Gr e~ka~tic oru~ . Such ~3=~ inclocb
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medicsl, ve~er~ary" and agricultural d~a~no~ics and
industrial ant pha~aceutical quallty cotltrol.
The invention involves a methot for u3ing
specifically producet nucleic acid~ complesllentary
S to, not only R~ and t-RNA, bu~ al o to ~pecific
~equences ar popula~ions of different sequences of
the RNA class mRNA or hnR~A. 09 STlEUlA or the l-La~ of
RNA sequence~ (hereinafter knolwn a~ precur~or ~pecific
RNA sequenceq or p8RN~) which are present only in the
precursor3 ~RNA, t RNA, hnRNA or ~n~aA molecules and
not in ma~ure mRNA, R-RNA, t-R~, hnRNA or ~nRNA
~lecules, to detect, identify and quantl~ate specific
organisTns, group or organism~, groups of eukaryotic
cell~ or viru3e in cell~, by the proc~3~ of nucleic
acid hybridizstion .
My invention and the novel~y, utilicy and unknown
ob~r$0usrles3 thereof can be more clearly u~der~tood
a~i;d ~eciated ~ ~i~et in th~ l~ght ~ ehe additional
. remesen~t~ve ~nd inform~tion hereinater set ou~,
2û comprislnE~ thi~ art;
1. n~A~, and psRNAs are preseYlt in all organi~m3
and c~ , hnRNAs a~d ~nRNAs ar~ present only in
eulcaryotlc: cells. Cell orga~ell~ which contain
DNA, i~clud~g mitochondria and chloropl~s 3,
al~o conta~n m~UaA, p~A, R-~, ~i t-RNA.
2. A typlcal bacterial cell con~ains ~Dore thara
a thousand gen~s, the ~a~ a~ orit~ of whlch
cod~ for a specl~ic prot2in. A m~malian c~ll
::: cQnt~ins over~lO,ûOQ genes e~Ph of which can
produce RN~. Any g~e ha~ ~he potent~al to
p~oduc~ ~ultiple cop~e~ of RNA in a cell. Each
specific RN~ olecule protuced :13 a direct gene
produce .
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3. Many dlferent mRNA ~equenoes can be
present in eaSh organls~ or cell. The
individual cells of a mul~icelled organiQm
may have different mRNA sequence~ pre3ent
in each s:ell or in different groups of cells.
Many differerle hn~NA, p RNA, and ~s~RNA
Yequence~ can be pre32nt in each cell or group
of cell~ of a eukaryotic organl~m. '.
Cells infected wlth a ~peciflc: v~ru3 can ..
have pre~ent withln the~D a varlety of dif~erent
typeY o viru~ speclfic mRNA and p~RNA
4. The number of copie~ l:hereinaf~er eha abundance)
of a 3pecific ~RNA in a prokaryotic cell varies
from z~ro to se~reral hundredr The abundance of
a ~peci~lc psRNA qequerlce ~ a prokaryotic
organi3~ or cell can be 10 to 20 tin~e3 hi~5her.
The abundance of a speclfic ~RNA ~olecule in
a eukar~rotic cell ran~es fro~ 1~2 ~o greater than
:; 10 per c~ll.
The abus~tance of a pecif ~ c hnRN~ ~equence in
a eukargotic cell range~ :~ro~ 1-2 eo greater than
10 pe~ cell.
abund~ce of a ~pec~fic s~A mol~cule in
a eukaryotic cell ~rarie5 fro~ 104 to 106 per cell.
The abu3l~ance o~ a specific p5RNl~ sequenc~ in
~; a ~ukar~otic cell var~e~ ~rom 1-2 to ove2 104 pe~
eell.
5. In ~an~r e~kar~otes, RP~ of ~variou~ type~ ~ ~
produced fro~ tlie repeated ~equence frac~ion~ of
: th~ 1)~. Thl~ can resul~ in a populat~on of
abu~dant R~ ~olecules who~e ~eque~ce8 are ~i~lar
bu~ not identical to one another. A probe co~le-
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meneary to one of ~hese RNA molecule~ wlll,
however, hybr,~d~ze w~ th all o the other
9imilar RNA molecule~.
6. Th~ gesle sequence~ which code for the various
individual mR~As. psRNAs, hr~7As and snRNA~ of
vi:c'u5e~i and living organiqmo, hav~ beell con~erved
to varylllg degreeY through evolution. The vast
ma~ority of thece sequence~ are much.le~s.cOnserved
than toRNA qequence~. SOme of the sequenc~s, h~wever,
are hlghly conservet. For exar~ple the . gene which
code~ for histon~ mRNA i~ very h~ ghly con~erved
through evolution and the hi~ton~ ger~e ~equence
is quite ~imilar irl w~dely differerl~ organisms.
l~he lack of conservae~on in th~ DNA 3e~uence3
1~ of ~any of the e RN~s all~w~ the production of
:~ probes whi ch can readlly distingui~h between
clo~ely rela~ed organ~ sm~ or ~iruse~.
A large number of s tudie s have been done on
var~ ous mRNA8, hlRN~g, snRNA~ and p~7A~ (see
Gene Expr~sios~ by B. ~ewln, in
r~fer~nces). Thes~ inciude hybridiz~tlon of these
RNAs i~ ~:ud~e~ ~n genetic analy~is ~ regulatio~
and evolution, ~n prokax~o~ and çul~aryotic
organ~ and viru e~.
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- 1 2 -
Prior Art Hvbrldization Procedures
Two basic nucleic acid hybrldization procedure~
are disclo~ed in the prior art. In one, in solution
hybridizatlon, both the probe and sa~ple nuole~ c acid
5 moleculeR are free in ~olution. With the other method
the ~ample i~ immobllized on a ~olid ~upport and the
probe i~ free in solution. Both o~ the~e methods are
w~dely u~ed and well documented in the literature. An
ex~le of ~ olucion m~ presented ~lereinaftOE ~I th~
10 eaE~sple~ o, in ~ article by ~ et al., Proc. ~atl. Acad.
Sc$. USA ~1980), 77, p. 520, i~ an ex~le of the im~bilized ~d.
.. _~v . ... .. . . ... . .
The basic co~pon~n~s of a nuclelc acld hybridizat~on
te t are:
1. Prob2 - A market ~ingle 3~saIad nucleic acid
~equence which i~ comp~ementary to
the nucleic acid qequences tG be
deeected (that i~ the tar~et sequen-
ce~). A~ u~ed herein, the target
sequQnce ~ ~ the ~cotal sequence or
a sub-~equence of R-RNA, t-RNA, or
other RNA.
, . .
The prob~ lengt~ ca~ vary fro~ S ba~e~ to ten~ of
thou~ant3 of b~e~, and will depend upon the specific
te~t eo be doll~. Only part of the p~obe ~olecule need
25 be complementar~ 'co ~he nucleie acid sequence to be
de~cected thereinafter the targe~ qequenc2s). In addition,
the comple~entarity between the prob~ abd the target
sequence ne~d not b~ perect. Hybrldi2atiosl does occur
be~een i~p~rfectly co~plementary ~olecule~ wie~ the
30 result thae a e~ri:ain fraction of the ba~e~ in the
h~bridized region are:no~ paired with the proper comple-
~n~ary ba~. A p~obe may.be compo~ed of either RNA
or DNA. The ~orn~ o th~ nucleic acid prabe 2~ be a
mar~ed sl~glQ ~trant T~lol~cule of 3ust one polarlty or
7~3~387
- 1 3 -
marked Qingl~ strand mol~cule having boeh polarit~eQ
pre~ent. ~he form of the probe, like ~t~ length, will
be determlned. by t~e t~pe o~ hybridization test to be
done .
2. Sample - The ~ple ~ay or may not contain
the targee molecule ( i . e . the
organi~ ~f intere~t). The sample
may take a variety of for~,
including liquid such as water or
seru~, or ~olld such as dust, ~oil
or tis3ue sa~ple3. The s'ample
nucleic acid must be made available
to contact the probe be~re any
hybridizatlon of probe and targat
molecule can occur. rhu~ the or-
ganisD~I 3 RNA must be free from the
cell and placet under the proper
corldielon~ before hybridiza~cion
can occux. Prior art method3 of
in solution hybrldiza~ion neces-
~Itate the pi~I~f the RNA
in order to be able to obtais
hybridization of the ample R~
~ with the probe. This ha~ mea~
:~ 25 that to utilize th~ in ~olution
method for det~ctinE~ targët
sequence3 in a ~a~ple, the nucleic
acid~ c~f the ~a~ple Dlu~e f iS~t be
purified to el~ina~e proteir~,
llpid~, and other cell component~,
and then con~cact~d with the probe
~: ~der hybridizati on condition~ . The
purlficatlon~ o~ the ~ample nucleic
acid take~ at lea~t several hours
and can take up to a day ~ dependir~g
~; : on the nature and quantity of the
sample .
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: 3 . Hybrid~ zat~orl
M~hod - Probe and ~a~ple fflU~'C be mixed lmder
~: 40 condition~ whieh will per~ nucleic
......... ... ............. ........ ... ... . ac~d hybrit~z.tio~ n~rol~re-~
con~acting th~ prob~ a~d 3ff~l~ in
the pre sence of an i~organic or
~` or~anic ~alt under the proper concen-
: eration arld te~pera~e condit~ on
prob2 and ample nucelic acid3
DW~ b~ ln contact for a long enough
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tlme that any possible hybrid-
ization beeween the probe and
~ample nucleic ae~t may occur.
The concentration of probe or target ~ the
mix~ure will determine the ti~e necessary for hybrid-
ization to occur. The hlgher the probe or target
concentration the ~horter the hybrldization lncubaeion
time ne~ded.
A nuclelc acid hybridlza~on incub~ation mixture
composed of pro~e and ~ample ~ucleic ac~ds mu~t be
incubated at a specific tempera~ure for a long enough
t$m~ for hybridization to occur. The lengeh o~ time
~ece~sary for hybridiza~ion to complete depend3 upon
the concentration of the probe nucleic acid, the
concentration o~ ~he ~ample nucleic acid which i~
; ~omple~entary to he probe, and a ba~ic rate of hybrid-
iza~ion wh~ch i~ ch~race~ri~tic of the hybridiza~ion
condieion~ u~ed. The ba~lc rate of hybridization is
~de~er~ined by th~ ~pe of ~alt pre~nt in ~he incubation
mlx, it~ concentxatio~, and th~ temperature of incubatio~.
Sodiu~ chloride, sodlu~ pho~pha~e and 30dlum citra~e are
: the sal~ mos~ frequen~ly u~t for hybr~tization and
~h~ ~alt conee~tration u~ed i8 rarely above 1 M and
:~o~e ime~ a~ high as 1.5 - 2 M. The salts mentioned
~25: abo~e yield co~arable ra~es of nucleic acid hybrldi-
~ ~ zatio~ when u~ed at the ~ame concentration~ and te~pera-
:~ hur~s, a~ do the co~parable po~as~ , lithiun, rubidiu~,
and CQ~iU~ ~alt~ Britte~ et al. ~1974~ (Method~ in
Enz~mology, Volu~e XXI~, pa~ E., ~d. Gro~sman aad
~olda~e; Acade~ic Pres8, N~w Y3rk, page 364~ and ~etmur
s~d David~on (1968) tJ. ~olecuLar Biology, Vol. 31, page
,
.
':
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gL;~78~387
- 1 5
349) present data wh~ch illustrate~ the 3tandard
basic rate~ of hybridization attained in commonly
used salt3. The hybridlzation rates of DNA with RNA
vary somewhat from ~ho~e of DNA hybridizing with DNA.
The magni~ude of the ~ariation i~ rarely over tenfold
and varie~, depending for example, on whether an excess
of DNA or RNA is used. See Galau et al. ~1977) (Proc.
Natl. Acad. Sci. USA,. Vol 74, ~, pg. 2306).
Cert~ln conditio~s re3ult in the acceleration of
DN~;~NA hybridization. An emul3ion of phenol and al
pro~otes the very rapid hybrid~zaeion of DN~ when the
~xture 1~ agitated. Rate inerea~es ~everal thou~and
ti~e~ fa~ er ~han standard DNA hybr~dizati4~ rate~ are
attalned with .hi~ sy3te~ (Xohn~ et al.> Biochemi~try
15. ~1977) Vol. 16, p. 532a). DNA hybridizaeion rate
acceleration fo 50 to 100 fold over the standard rate~
haY al~o been ob~erYed when neutral and anionic dextran
polymers w~r~ ~ixed with slngle ~trand DNA in ~olution
(Wetmur, B~opolym~rs (1975) Vol 14.-p. 2517). Nei~her
:~ 20 of the~e DNA ace~leratet rate condtion wa$ reporter ~o
accelerate the hybridlzatio~ ra~ of DNA:RNA hybridi-
z~tlon~ no~ a~are of any prior art which documents
a condltion for accelerating th~ r~te of RNA:D~A
hybritiza~ o~
4. ~ybrid~zation
A~a~ - A procedure i3 need to detect ~he
pre~enc~ of probe molecules
hybridized to th~ ta~g2t molec~le-~.
- ~ Such a ~ethod depend~ upon the
abilit~r to ~eparate probe which 19
h~ridiz~d to targ~t molecule~ fro~
prob~ whlch i~ not hybrid~zed to
~arge~ ~olecule~. Prior are pro-
cedure~ or a~sa~ g in ~olu~ion
hybridization D~ cures ha~i
done on ~ample nuGleic ~cids which
ar~ st purified a~d then oontact~t
wlth the pro~e in the hybridization
Lncutl~t lo= mi~cturl! .
;~
.
~.
.
~L~7~9~37
-16-
Hydroxyapatite (~) h~s been u~ed a~ a standard
method for a~ayln~; in ~o~ution hybridization mixtures
for ehe presenee o~ hybridized probe. Under the proper
conditions HA selectively bind~ hybridized DNA probe
5 but does not blnd probe which is not hybridizPd. Other
methods are available to a~say for hybridized probe.
The~e include Sl nuclease as3ay wh~ch depend~ on the
ability of a ~pecific enzyme to degrade non-hybridized
prob~ to small subunit~ while the hybridizet probe i not
10 degrad~d by the enz~e and remains large. Th~ ~cgraded
probe can the~ be ~eparated from the hybridized probe
by a ~ ze separation eechllique . Variou~ method3 for
a~saying for in soluelon hybridized nucleic acid3 are
pres~nted in Britten ~e al. (1974) ~upra.
The immobili::e~ ~ample nucleic acit hybridization
method~ haYe the hybridization assay bullt lnto the
hybridization ~ethsd. The~e method inYolve fixing
th~ 3amp1e nucleic: ac~d onto an iner~ c~upport and t'nen
h~bridizing thi~ i~nmobilized nucleic acid with a marked
probe which i~ free i~ solutlon. Xybridization of any
probe with the i~mobilized ~ample ~ucleic acit results
th~ bi~din8 of the prob~ to ~he ~a~npel nucleic acid
~d ~here~ore the attach~Qe~t of ehe probe eo the iner~
support. N~n~hybr~d~zed probe rema~Lns free ~n solu~ion
~: 25 and can be ~ hed away fro~ th~ inert ~upport and che
hybritized probe. Such a method requ~re~ at least
~everal hours to prepar~ th~ sample for nucleic acid
hybridizatios~ and one to two hour3 of wa3hing and
utilizeq large a~o~t3 of probe~ An advar~tage of ~hi~
E~thod is the capab~lity to place multipl ~ ~ample~ on
~: th~ ~e lner~ suppor~ and to hybFidlz~ and process all
th~ le~ a~ os~e time. Examples or' ~uch an im~obilized
3a~1e ~ethot i~ pr2s~nted i.n Analytical Biochemi~ry
~ ;~78'3
-17^
(1983j Vol~ 128, p. 415, ant J. of Infectlous Disease
(1982~ Vol. 145, ~,6, p. ~63.
Ma~cing Nucleic Acid~ Available for HYbridization
In ~ nuclelc ac~d hybridization methods
5 have alwayq utilized nuclelc ~cids which have been
purifled away from other cell components. Nucleic
acid3 in cell~ and viru ~ are normally tightly ~
co~plexed wlth other cell componen~, uQually p~otein,
and, in thi~ form are llot available for hybridi~st~on.
~0 Simply breaking ~he cell or v~rus open to releas~ the
cont~nts does not r~nt~r the nucleic acid~ available
for hybridization. The nucleic aci.d~ remain coslplexed
to other cell or viral components even though rele~ed
fro~ the cell, and ~ay in fact beco~e exten~ively
15 degraded by nucleQses which also maybe relea~d. In
addition a mar~ced probe added to such a mix may beeome
complexed to "~tirky" cell or ~iral components and be
rendered ~navailable f'or hybridiza~cion, or the probe
may be degraded b r nuclease action.
A variety o prior are ~thod exi3t for purifiying
nucleic acld~ ant se~eral of these are described in
Maniati~ et ~1., 8upra. These method~ are all time
consu~ing - ~one talking an hour i~ re8arded as very rap ld- -
and require ~ltiple manipulation~.
. Insofar as I a~ aware, there is no pr~or art method
or performlng in ~ ution nucleic acid hybridization
which doe~ not . require the use of ~ome sort of prepuri-
ficat~on s~ep ~o^~lke the nucleic ac~d~ available for
hybridlzatio~ .
The i~aoblliz@d mlcleic acid hybridlza~ion methods
~avolve fi~inl3 t~e ~ ple nucleic ac~d onto an inert
suppor~ and then hybridizing thi~ imeaobilized nucleic
~.~
~.~78~387
-18-
acid with marked probe whieh is free in solution.
The proces3 of flxin& ~he nucle~c acid~ on ~he intert
support provid2s a pur~fication 3tep effectlve enough
to make the bound nucleic acid~ available for hybridl-
5 zatlon. Mo~t of the non-nucleic acid cell or viral
componen~c3 do ns~t bind to the iner~ support, and those
which do bind do ~o at a different location than the
nucleic acid~. Such a method require~ several hours,
at a minimum, to prepare ~he sample nucleic acid for
hybrid~zatlon. An advantage of thi~ method i9 the
ability to place ~ul~iple ~a~ple~ on the i~er~ 3upport
and proce3~ the~ all tcgether through the hybridization
and ehe hybrldizatio~ a~say step~. The hybridization
a~say con~t~ of remo~in8 the inert ~uppore from the
hybridlzation m~xture. Prove which i~ hybridized to
the fixed 3ample re~ain~ with the intert ~upport while
non-hybridized probe remain~ free in ~olution.
`~
:
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`:
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31 ~7~9~7
-19-
TFIuq, while the pre3ence of organlsms can be
detected by any one of a large variety of prior art
method~, none s~f the~e 1~ entirely satiqfactory for one
rea.~on or another. Such method~ include , e . g., growth
5 n:ethot~J opeical detection methodQ, ~erolog~c and
im~nunoche2~ical method~, and biochen~ical method~, as shown
below: -
Growth Tes t ~:
A large number of different growth te . t~ exis~,
lQ each useful for the growth of a speciflc organi~m
or group of organism~ . G:rowth tese3 ha~ e th~ potential
~ensitivi~y to detect one organi Dl. In practice, however,
many organls~ are difr'icul~ or impo~ble to grow. These
te~t are usually lengthy, taking fro~ one day, to ~on~h~,
15 eo co~2plet e. In add~tion, ~ ~ery large n~ber of tests
would be needed to detect the pre~ence of any me~ber of
a large group of organis~s ~e . g ., all bacteria~, assuming
that the grow~h conditions for all members of the ~3roup
are kxlown.
20 OpticaLl Detectioa Method~:
M1 cro~copic aaaly is coupled wlth different~al ~t~ining
~ethod~ i~ very po~7~r~ul, and in znany ca~es, ver~ rapid
detectio~ ~!thodO A ma~or problem w~th thi~ approach
i~ the derect~on of specific organl~ in the preQence
25 o~ large quantltie~ of other organi3ms, for e~cample, the
iderltificstlon of a 3pecifle typ~ of graTn negative rod
~haped bac~er~a, irl ~he pre~nce of many d~fferent kindQ
of gra2ll nega~ive rod shaped bactaria. In addi~cion, a
large number of te9t~ would be needed ~o det~ct the0 presence of ~11 me~nbe~s of a large group of osganis~ns
ch a8 the group o~ alI bacteria).
.
- 20~ 789~7
Serolog~c and I~munochemical
Me~hods and Biochemical Te~ t 9 -
_ _ _
A larg number of different types of the3e te~ts
exist. They are usually qualieative, not very se~3itive
ant often require a growth step. A great many of these
teqts would be required to detect all members of a large
group of organi~m~.
* * * * *
U.S. Paten~ 4,358,535 to Falkow et al. di~clo~es
a m~thod for the detection of genet~.c material, l.e.,
G2ne~ or Genome~. In this patent a clinical sample or
~solate su~pect~d of containlng a pathogen is transferred
onto an inert pvrou~ Yupport, ~uch a~ a nitrocellulose
fllter, and treated in ~uch a way that ehe cell~ are
localized. The cell~ are th~n treated ~n such a way
as to release their DNA and cau~e it to eoupl~ on~o
the suppor~. Subsequent treatmen~ cau~e~ a separa~ion
of the individual DNA ~trand~ of the geno~e. The strand~
are then contac~ed with labeled prob~ ~peciflc for the
characterl~tlc polynucleotide ~equence under hybridiza~ion
condltion~. Hybridiza~ion of the probe to the ~ingl~
~ ~tranded polynucleotide~ fro~ he pathogen i~ de~ected
: by mean~ of ~he lab~l.
The method of ~hi3 paten , for detectlng gene~ or
g~nom~ ke ~h~ oth~r m~od~ ~ntio~ed abo~ does not
have the ~pecif~city, sen~itiYlty, rapidity or ea3e o~
performanee o t~a~.o~ ~y ~n~en~ion. A sum~2ry of
com~ari~on~ o the Falkow et al. m~thod as di~clo~ed in
~he pa~ent and tha~ of applicant ' ~ method, a~ h~rein
dlsclo~ed, i~ ~et out below:
;~
.:
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`~
~.2~89~37
- 2 1
1. Method of doing hybridization
FAI~W ET AL. METHGD APPLICANT'S METHOD
Im~obilized ~ethod only In Solution ~ethod emphasi ed.
Im00bilized method can be used.
2. Class of nucleic acid to
be detected
FAI~~W ET AL. METHDD APPLICANT'S METH~D
,
Geneeic ~aterial (i.e., Detection of a primary gene
Genes or Genom s). In product (RNO cnly. RNA is
cellular organisms the not present as genetic ~sterial
genetic ~aterial is always in cellul~r organisms.
D~A.
3. Abundance (copies per cell)
of nucleic acid sequences
to be detected
FAI~ ET AL. ~T~D A~PLICANT'S MEl~lDD
Vi~tually all microorganism ~ of R-RNA are pres~nt
chranosc~al genes ~e pre- per bactcrial cell. A~out 2 x
~: ~ se~t anly one ti~la per cell. 133 capies of each t-R~ is
rouDscmal gene are present lsl each bacterial cell.
ususall~ presene 1-3 time~ Ten to 200 of each specific
1~. Ribosa~al RNA ~IA molecule is present
are pre~ent 3-6 ti~ p~ bac~erial cell. The
: ~ per cell. ~ are ger~ally hi~her
in e~cæyo~c cells.
,: ~
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4. Ability of hybridization ~ethod
to quantitate nucleic acids
FALK~W ET AL. MEIHDD APPLICANT'S METHOD
None disclosed Excellent ability to quantitate
nucleic acids, both DNA and
RNA.
5. Ability to determine and quantita~e
the state of genetic expression of
a cell
F ~ ET AL. ME~ D APPIICANT'S MEXXOD
Genetic expression cannot be Genetic expre.qsiQn c~n be deter-
detersined by deteeting ~Qncd and quantitaeed by usLng
genetic material. probe~ ~ich detect the pr
gene pr~ducts or RN~s.
~: 15 6. Relative probability of detecting
a false positi~e during diagnosis
FALKOW ET AL. ME~H~D APPLICANT'S hETHDD
High (deteets only specific Low ~*~n emphasis is ~n
genes). detec~ing RNA~.
: ~ .
20 7. Relative sensiti~ity of detection
of n~cleic acid~
ALI~W El ~L. ~ {~3 APPLICANT'S METHDD
Good. Nuclei~ acid hy~ridi- Hi ~ y sen~itive. From 20
za~ion test axe ln general to 104 times more sensitive
~: ~ 25 ~ qui~e sensitive. than possible with the approach
outlined i~ Falkcw. RNA is almost
;~ alway~ mDre abundant than the genes
: : which ~akR i~. The in solutisn
ne~hod also ccnfer~ extra
,.,
~ 3a ~ sensitivity oNer th~ imIobilized
,
~ :
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-23~ 3~7
8. Pre3aration of s~le for
~ybri~izatian te~t
F~ Er AL. MEl~D APPLICANr'S ~ DD
Takes fr~ 2 - 10 h~s co Takes 1 - 5 minuteS to make
im~bilize sallple rTucleic sa~le available for hybridi-
acids and ~ake th~ avail- zation. RNA in cells is alrea~y
able for hybridizatiasl. singl~ stranded. All of the
Includies a step for corl- s~le ~ucleic acid is capable
verting D~A to si~le strand of hybrid~zing.
form. Not all the sarrple
T~cleic a~ ~e capable of
l~ybridization .
9. h~nt of pro~e needet
F~ E'r AL. ~ APPLICANI 'S ~ET~D
Us~lly taked 0.01 ~o 1 Need 10 ~to 10 6 micrograms of
microgra~ of probe in probe per sa~l~.
~ybridization m~cture.
10. Ti~e needed far ~ybridization
to oc
~; 20 FA~W ET AL. ~l~W APPLICANI'S MET~DD
2 - 20 h~s 0.2 - 0.6 hours
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1~ 7~7
-24
I am not aware of any prior art which teaches my
method of detectin~ th2 presence or ab~ence of R-RNA,
or of e-RNA characteristics of a particular group of
organism~ utilizing nucleic ac~d hybrid~zation wherein
i~ used a selected mearked nucleic acid molecule comple-
mentary to a subsequence of R-RNA from a particular
source. Nor am I aware of any prior art which discloses
my method for detect~ng the presence or absence ~ R-RNA
in general, or of ~-RNA fro~ a particular source, by
19 nucleic acid hybridiza~ion u~ing a marked nucleic acid
molecule complementary to all of ~he R-RNA, or t-RNA
subsequence fro~ a specific sourc~.
Nor a~ I aware of any prior art whi~h teache3 my
~ethod of detectlng the pre~ence or ab~ence o~ specific
; 15 ~equences or populations of different Qpecific sequences
~:~ of mR~A, psRNA, hnRNA or sn~NA to detect, identify and
quantitate specific organis~, group~ of organis~ groups
: of eukaryoti cell~, or specific viru~e~ in cell~ or a
group of speeific viru~e~ i~ cells, by nulceic acid
: 20 hybridization wherein ~ u~ed selected marked nucleic
acid molecule~ com~le~entary-to a sub~equence(~), a
~equence~ or ~ population of ~equence~ or ~ubsequence3
`~: of ~R~A, hnR~A, ~nR~A or p~RNA fro~ a particular source.
Nor ~ I awar~ of any prior art which teaches my
~ethod of detec~ing the pre~ence or a~sence of a nucleic
~; acid charac~eri~tic o~ a particular group of organis~
: or virus~; or of rap~dly m2kl~g a~ailable foriA solution
nucleic acid hybrtdization wi~h a spec~fic marked
:~ ~ probe, the nucle$~ a~ids of a partleular group of organisms
: 3Q or viruses for any purpo~e; or of utilizing ~n in solution
~: nucleic acid hybridization method which eombine~ a rapid
'
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,.
7~3~7
- 2 5 -
me~hod for making the nucleic acids of specific groups
of organi~ms available for hybridization with a specific
complementary probe, with a method for detecting an
or~anls~ nucleic acid by greatly accelleraeing the
5 rate of in solutlon hybridiza~ion of the nucleic acids
of an organi~ or viru~ and the marked probe complementary
to ~he organism' ~ or virus ' ~ nucleic c~d; or of
determining the anti.microbial agent sen~itivity or
an~iviral agent sen~itivity of a particular group of
10 organis~n~ or viru~e~; or of assaying for the prëQence
of antiDIicrob~al or antiviral sub~tance~ in bloot, urine,
other body ~lu~ds or ti~sue~ or other qa~ple~; or for
dete~Qining the sta~e of growth of cell~; or of detecting
microorganisDI or viru3 infection-~; or of rap~dly assaying
l~S for the presence, ~n a hybridizaeion ~nixture, of probe
which has hybridized, by contas:ting the Dlixture with
hydroxyapatite u~der pred~t~nined conditions and ther~
processing the re-culting solution in a 3pecific manner.
.
DISCLOSURE OF THE INVENT~O~
29 The pr~sent in~ention provide~ a method and mean~
for detec in8 3 identifying, ant quantitatin3 organis~a~
1~ blological and oeher sampl2R, and ~ore particularly
to a m~thod for ~peclfically and ~en~itively de~ecting
and quanti~cs1:ing any organi~m containing ehe ribo~oma~-
RN~, ~hereinafter R-RNA) ~ ~ran~fer RNA (hereinafter
t-RNA~ or other RN~; any me~ber~ vf large, intermed~ate,
or ~11 sized categories or taxonomic group~ of such
organl~c; and previously unkno~ organ~ms containing
R-RNA or t-}WA. The ~ethod i3 eapable of detecting ehe
:
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~L~7~{3~7
-26-
presence of ~ven one organis~, containing R-RNA or
t-RN~.
The invention al.qo provide~ a method for using
specifically produced nucleic acids com~lementary to
specific sequences or pop~lation~ of different sequences
of the RNA cla ~ mRNA, or hnRNA, or nRNA, or the class
of RNA sequence~ (hereinafter precur~or specific RNA
sequences or p~RNA) which are present only in the pre-
cursor mRNA, R-~NA, ~-RNA, hnRNA or snRNA molec~le~,
and not in matUrQ m~NA, t-RNA, hnRNA or snRNA molecuie
~o deeect, ldentify, and quantitate speclfic organisms,
~roups of organi-Rm~, groups or eukaryotlc cells or
viru~e~ in cells.
~he ~m~tion also pro~ a me ~ d 2nd.~ h~n~ ~ ~t~-
~z~n~ itie~: (a~ ~.abilit~ ~o ~cifically dbtecc ~ Presen~:e
of an~ ane of a læge ~er of diffsr~t or~ wil:h a ~ingle assa~r
procedure which al o works regardless of the l?attern
: ~ of geneeic e2pres~iorl o~ any particular organiQm; ~b)
~: 20 the ability to ~odify the test to detect only specific
cat~gories of organi~m~ 9 even in the pre~enee of organisms
noe in ~he group of intere~t, tc~ extremely hi&h sensitivlty
of deteetion9 ant ability to detect the presence of one
organ~ or cell; (d~ the ability to quan~itate the
numb~r of organis~ or cell~ present; and (e) doe~ not
require a gro~th s c~p .
My invention proYide~ means for detecting the anti-
mi~robial agent en~i~ivity or antivlral agent sens~ ti~i~y
of a particular group of or~an~sms or viru~es; for assaying
30 the pre~nc~ o~ an~ crobila or antiviral $ubQtsrlees in
,~ .
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789
-27-
blood, urine, o~ ~r fluids or tissues c~ other sa~les; for
determining the state of growth of cellR; for deteceing
microorganlsm or viru~ infections; and for rapidly
assaying for the presence ln a Xy~ridiza~cion mixture
5 of probe which ha3 hybridized.
As described hereinbefore, R-RNA base sequence~ are
partially similar in widely different organisms. The
more clo9ely related two organis~ are, the larger the
fraction of the total R-RNA which is ~imilar in the two
10 specie~. Tha ~-RNA sequence of any par~icular pecie~
or orgarll~m can be regarded a3 a serie~ of ~hort R-RNA
subse~uenceY, of which ~ne sub~quence 1~ 3im~1ar in
vir~u~Llly all life r'or~. Therefore, the R-RNA of
almo~t all life for~ t contain thi~ ~ub3equence.
15 A different subqequence i~ q~milar only in the R-RNA
~f the members of the Species ~o whi ch that organi~m
belongs. Other subsequences are present in ~he Order
or orga~ism~ thac the Specie~ belongs ~o, and so on.
Becau~e th~ R-RNA sequences of widely t$fferent
20 organis~ are a~ lea~t partially . imilar, the ~nethod
of my inv~ntion, u3ing a proba which d~tect3 the R-RNA
sequence~ which are imilar in widel~r tif~erent crganis~as,
can tet~c~c the pre3e~ce or ab~ence of an~ one or mor~ of
those organism~ in a sample. ~ ~ar}ced nucleic acid
25 sequence, or 3equences oomplemen~cary to ~he R-~IA
~equence3 ~imilar in widely di~,rergent organism~, can
be uced as such a probe in nucleic acid hybridization
assay .
Because of the R-RNA sequences of closely related
30 organis~n~ are more si~ilar than t~o e of d$~caIltly related
organism~, the ulethod of my in~ention, which includes
u~:Lng 5 probe whlch detect9 only the R-RNA sequeLces
. ..
~7~87
-2~-
which are si~ilar in a part~cular narrow group or
organi~m~, can detçct the pre~ence or ab ence of any
one or more of those particular organis~ in a sample9
even in the pre~ence of many non-relatet organisms.
These group specific probes can be ~pecific for a
variety of different ~ized cat~gories. One probe might
be speclfic for a particular taxonomic Genu~, while
ano~her is specific for a particular Family or a~other
Genu~.
Group specif~c probes ha~e the ability to hybridize
to the R-RNA of one group or organism~ but not hybridize
to the R-RNA of any oth~r group of organ~sms. Such a
group ~pecific eomplementary ~equence will deeect the
pre~ence ~f R-RNA from any me~ber of that spe~ific group
of organi~ms even in the presence of a large amoune of
R~RNA from many organlsm~ not belonging to that specifl~
group.
The total number of R-RNA molecule~ in a 3ample i~
~easured by u~ing a marked ~equence or sAquences
co~ple~en~ary to R-RNA and ctandard exce~s probe or
exce3s sample ~NA nucleic acid hybridization methodology.
Th~ R-R~A con ent of cells fro~ a wide variety of
organi3m~ i~ known in the ~rt. In a broad group of
~imilar organi~, fo~ example bacteria, the amount of
R-RNA per cell ~arie~ roughly 2-5 fold. Therefore, if
~ th~ number of R- ~ A ~oleculeQ in a ~ampley and the broad
- : . class iden~ty of the source of the R-RNA i~ known,
ehen a good e~timate of the number of cell~ pre ene in
the ~am~le can be calculated. If th~ broad cla~
lten~ty ~9 not known it can be de~er~ined by hybrid~zing
the ~ample eo a serie3 of seleotet probeQ com~le~en~ary
~o R-RNA, each of which lx 3pecific for a par~ieular
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broad category of organisms.
At the presen~ ti~e, the operation~l detectlon
and quantitation rang~ of a single a~ay procedure i3
fro~ 104 R-RNA ~olecule~ (1 bacterium or 10 2 mammailan
cell.~) to abou~ 1012 R-~NA ~olecule~ (108 bacteria or
106 mammalian cellR) a ~pan of about 108 in cell numbers.
A single te~t could also be don~ in such a way a~ to
operaeionall~ quantiea~e from 103 bacteria to 1~1
bacteria. The t~t is quite flexible ~n thi~ w2y.
Because the test for R-RNA i~ specif~c an'd has the
ability to detec~ the presenee of ~ery few organlsms
there is no need to ampliPy t~e numbers of organisms
through a growth step.
The practice of ~hat orm of my invention which i4
directed to determining the presence o~ ~n organi~
whlch contain~ R-RNA, in a sample which ~ight contain
uch organis~, comprises basically:
a) bringing together the sa~ple, or isolated
nucleic ac~dq contained in that ~ampl~, wlth a probe
~0 wh~eh co~p~i~e~ mark~d nucleie aeid ~olecule which
.; ar~ comple~entary to he R-RNA of all organism~;
b) incu~atin~ ~he se~ul~ing ~ixtur~ under pre-
deter~ined hybr~dizaelon conditions for a pr~teter~i~ed
e, a~d th~n;
c) a~aying the resulting mixture for hybridization
of ~he probe.
~: When ~ in~entio~ i~ directed to determlning the
~ . presence of any me~ber o a speciie catego~y of organisms
.~ ~hlch contain R-RNX in a ~a~ple which migh~ con~ain cUch
~ 30 organls~ ~ ehe m~ hod compri~es:
,;,
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-30-
a) contacting the sample, or the nucleic acids
therein, with a probe compri~ing marked nuclele acid
molecule.~ which sre complementary only to the R-RNA
of meDlbers of the specific category or organis~s, but
5 no.t complemen~ary to R-RNA from non-related organism~;
b) incubatinE~ the probe and the sampl2, or the
i301ated nu~ leic acid~ therein; ant
c~ as~aying the incubated mixture for hybri~ization
of said probe.
My inven~ion can lso be used to deterr~ine the
nuDlber of organism~ pre~eat in th~ ~ampl~ under investi-
gation, bg adding to the as~ying in the second above
da~cribed method in the event probe hy~ridization has
occurred, ~he s~ep of comparing the quantity of R-RNA
15 present in the ~ample with the numb~r of R-RNA molecules
nonnally present in individual orgarli~s belonging eo ehe
said ~peci~ic group.
And, of course, included in the Yariations ~ within
the Ccope of D:~ invention ~ which can be u~ed, i~ that
20 which co~pri~es, in lieu of the single pro~e o~ step
~a) in the 3econd ~f the above methot~, a multipl~ city
or ba~tery, of di.fferent probe~. In such case, each
~eparaP~ prob~ co~pri~es marked nucleic aeid molecules
which are co~pleme~ta~y only to the R-RNA of a ~pecif i~
25 group of organl^~n~s and each probe i9 ~pecific for a
different g~oup of organism3; ~tep (a) i~ followed by
i~cubating e~ch probe-~a~rple ~ixtur~ under predete~mlned
hybridizatioTI eondit~on~ for a pre~d~ter~rled time, and
then assaying each ~ixture for hybridization of ~he probe.
:~:
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-31-
As descrlbed hereinbefore t-RN~ base ~equences
are partially ~imilar in widely different organis~.
~he more closely related two organix~s are the larger
the fraction o~ t-RNA ~equence~ whlch are related.
Each t-RNA gene ~equence can be regard~d as a ~eries
of short t-RNA subsequence~. One ~ub~equence is similar
in a large related group of organisms. Another sub-
sequence is si~ilar ln an in~ermediate sized related
group of organism~ while a third qubsequence i3 similar
10 in a small related group of organi~ms and ~o on.
Since, alqo, ~ t-RNA sequence~ of widely different
organi~s are at least pareially similar, the method of
my invention, u ing a probe which det~ct~ the t-~NA
sequences which are simllar in widely dif~erent organis~,
lS can detect the presenee or absence of any one or ~ore of
tho3e organis~ in a sample. Thu3, a marked nu leic
acit sequence, or se~uence~ comple~entary to ~he t-RNA
sequence~ similar in widely divergent organism~, can be
u~ed as ~urh a probe in a nucleic acid h~bridization
a~ay.
And 3ince the e-RNA sequences o clo~ely rela~ed
organi-~s ar~ ~or~ -~imilar than tho~e of di~anely related
organi~m~, the ~æ~hod of my invention, whic~ includes
u3ing a probe whieh de~ec~ only the t-RNA sequences
25 whlch are ~i~$1ar in a particular narrow group of
organi~ can d@tec~c the pre~ence or abRance of any
one or ~nore of those particular organlsms in a sa~ple,
even in th~ pre~erlce of many non-rela~ed organisms.
Such group ~peci~ probrs can be ~p~riflc for a varlet~
30 of differene ~ized categorie~ . For example p one probe
:,
`:
89
-32-
mlght be speclfic for a particular ~a~onomic Genus,
while ano~her ~3 ~pecific for a par~icular Fa~ily or
another Genus.
Group specific probe have the ability to hybridize
S to the t-RNA of one group of organi~s but not hybridize
to the t-~tA of any other group of organi~ms. Such a
group specif ic complementary ~equence w~ll detect the
pre ence of t~ A fro~a any ~ember of ehat specifi~ group
of organi~ms even ~n the presence of a large amount of
t-PJM from rrlany org~ni~m~ not belong~ng to rhat specific
group .
In the pract~Ye of that forln of the inveneion which
iq direc~ced to deeennining the pre~enee of any me~ber of
a specific category of organi~ which contaln e-RNA In
a ~ample which might contain ~uch organi~aG, the method
compr i.~e ~:
a) contact~n~ the saDIple, or he mlcleic acid~
the~rein, with a probe compris~ng marked nucleic acid
molecule~ which are co~pleDlentary only ~o the e-RNA
of meD~ber~ of the speci~ic category of organism~, bu
not co~ple~ntary to t-R~A fro~ non-related or~anisms.
b ) incubating th~ probe and th~ le, or the
i~olaeed r~iucleic acid~ therein; anid
C) a388yi~ig the incubated mixture for hybridizationi
:~ 25 of -qaid prob~.
~r in~ention c~i also be used to det~rsaine the numiber
. of organi~m~ pr~ene in the ~a~plg under in~7estigation~ by
id~lng to the as~ing ~n the secont abo~re d2scribed methodi
in the e~en~c probe ~y~ridizatiorli ha~ occurred, the step
of c0~2iring t}~ie quanitity of t-RNA pre~ent in the sample
':
':
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~1 2789~37
-33-
with the number of t-RNA ~olecule~ normally present in
indi~ridual organi9Dl8 belonging to the said speoific group.
And, of courqe, included ln the variations, withi~
the scope of my invention, which can be used, is that
which comprlses, in lieu o the ~ingle probe of qtep
(a) in the second of ' the above methods~ a multipllcity
or battery~ of diferent probe~. In ~uch caqe, each
~eparate probe comprise marlced nuele~c acid moleculeq
whlch are comple~en~ary only to the t-RNA of a specific
group o organl~ms and each probe i~ ~pecif i c fo~ a
different 3roup of organi~; 3tep ~a) is followed by
~ncubatlng eaeh probe-~ample mixture under predetermined
hybridization condie~ on~ for a pre-determined time, and
then a3~ayln3 each mixture for hybridlza~ion cf th~ prob~.
The method and measn of ~y invention are Dlor~ fully
illustrated in the following descrlp~ion of characterizing
features of te3e method~ ~n aceordance with the in~entionO
Nuclei~ Acid Hybr~dization Test Procedures
A desirable de~ectlo~ te~ ~houldo a) be rapidi
b) be easr to U9~!i C) be highly se~a~itlve; d) be able
to detect and quantl~ate in ~u~ one lab a~ay.
The exiqtent of a nucleic acid probe wh~ch will
ybridize 'co R-RNA from any me~ber of the Genu~ Legionella,
but d~es not h~bridize to R~RNA rom any other ource,
make~ posslble a rap~d, ea~y ec, u~e, sensitive, in solu~ n
deteetion test which can bo~h de~ect and quantita~ce, for
exampl2, ~ bacseria with th~ performa~ee of just
orae laboratory a~ay and do~-Q not require the purification
of Aucl~iG ac~d8 fro~ the ~a~le.
''
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~1 ~7~3987
-34-
A description of the ba~ic aspect~ of thi~ in
~olution hybrid~zation te~t procedure ollow~. While
the procedur~ te.~cr~bed i~ designed for detecting members
of the Ge~au~ Le~ionella, it iq ob~r~ ou~ that this same
te~t procedure can be used with the appropria~ce probe
to detect many other group~ or organism~ or viruses.
Step 1. Preparine the S~ e
M~x the ample wi~h a solution containing a detergent
and a protealy~ic enzyme. The detergent ly es rhe baceeria
and help~ solubilize cellular com~onent~ whil~ ehe enzyme
destroys ~he cellular prote~ns, includ~ng ~hose enzymes
which d~8rade RNA a~d DNAo The com~o~ition of the
detergent-enzyme ~lx depends upon the type of detergent
: and proteolytic enzyme u~d ant the amount and type of
sa~Gple to be check~d. Detergent~ used include ~odium
lauryl sulfate, -~arkosyl and Zwit~ergent, while the
enzym~s u~et lIlclude Proteinase K and Prona~e. A wide
variety of en:zy~e~, -qolubilizing agents such as chao~cropic
: agent~, can be u~ed. The probe can al o be pr~3ent in
the detergent-e~zyme ~ix added to the sample.
The enzy~-~detergent act~ v~ry quickly on any
Lep~ion~lla bacteria in th~ ple. Irl lao~t case~ it
i~ not nece~ar~r to incuba~e the mix ure in order to
make ehe R-RNA available ~or in solu~lon hybrid~zation
wi~h ehe probe. In eertain ca~e~ a short incubation
perind i9 neet~d.
In other ~ituations it 1~ noc neee~ary to include
the proteolgtic enz~e, and de~cergent alone w~ll make ~che
~- R-RNA a~ailable for in ~olution hybridization with the
~`~ probe ~
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789~7
-35-
This approsc~ pro~tides a very rapid and easy method
for gett~ng the sa~le R-RNA into a ~tate where it can
hybridize with the probe is~ an in Qolutlon a~ay. In
addition, it allow~ ~he hybritization to occur in olution
5 w~thout purifying the qample R-RNA. A key to this method
that the probe detect~ Le~ionella R-RNA. R~
~ingle ~trand~d in the cell and reaty to hybridlze with
the probe onc~ the ribosomal protein3 are remove~ fro~
the R-RNA. In con~cra t, ~o directly detec~ the ribosomal
10 R-RNA DNA (l.e., the gene for R-RNA) or any other DNA
sequence it would be nece~sary to add a procedure which
cau~ed the double ~tra~ded R RNA gene to ~parate ln~o
two ~lngl~ strsnds befor~ the probe could hybridize to
~t .
To the best of my knowledge, there i9 no prior art
conc~rning the u~e of ~ enzyrn~-detergene-~ample method
for making R~RNA, transfer R-RNA, RNA in general or DNA
available for in solue~on hybridization ~ith a probe for
ehe purpc: ~e of detec~ing and quantita~ing the presence or
20 ab~ence of organ1-qm~ in general or a ~pecific group of
organi~m~ .
Step 2. Pre~srin~ the HYbridizatlon Incubation Mixture
To the sa~le-en~y~e-detergent miac add the probe
and sufflcient 3al~ to enable hybrid1zation to occur
25 and incubat~ 'ch~ resultant mixture at a~ appropriate
~emperature. The ~alt ccnce~tration and the temperatu~e
of hybridizatlon incubatisn combine tc determine the
cri~e2 ion. The crlterion of the incubation contition
mu~t be equal to that used to ~elect tha probe or the
30 ~pecifiri~y o~ the probe may change.
.,
~ 7~ 9
-3~-
~ e incubation mi~ture mu~t be incubated for a
long enough tlme fpr hybridlzation to oecur. The salt
typ~ and concen~ra lon determine~ eh~ rate of hybridlzation
which can be attained. Thu~ certain salts will promote
~ery rapid hybrid~zation when u3et at the proper concen-
eratiOn. An example of ~uch a salt i3 sodium pho~phate.
Legionella ~pecific probe mixed with purlfied Le~ionella
R-RNA in 3.0 M ~odium pho~phate buffer tpH - 6.8~
(hereinafter ter~ed PB) and incubated at 76 C hybridizes
la over 100 time-~ more rapldly ehan the ~am~ amounts of
Le~ionella prob~ and R-RNA incubated u~der ~tantard
cont~tlon~ of 0.72 M NaCL, 76 C (~he~e two condltiona
are equal in crieerlon). Other sal~ ean al50 be uset to
effec~ ~his hybridization rate acceleration. The~e include
~ost sodiu~, a~mo~ium, ru~idium, pota~ium, ceslum, ant
lithlum salt~.
. In 3 M PB at 76~ C the hybr~dization r~te of ehe
spec~fic probe wlth Le~ionella R-RNA present
in the PB-enzy~e-detergent-~ample probe mixture ~s
al~o sccelerated by o~er 100 time~ o~er the hybridization
rate~ seen for the standard incubation conditions.
:~ Hybridization al30 occurs between the proba a~d R-RNA in
an enzyme-detergen~-~ample mixture under sea~dard ~alt
concentration condltion~.
: 25 One o~ the feature~ of the invention, a.~ previously
poi~ted out, iq th~ ab~lity to detect very ~mall nu~bers
o~ organi3~s by detecting their R-RNA. Thi5 i~ po~sible
because of ~he large number~ of R-R~A molecule~ in each
; cell. Xn ~g~ like organisms 5,000 to 10,000 R-RNA
~olecule are present in each ~ndividual bacterial cell.
:~ :
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_37~ 78 98 7
One of the major determlnants of the ~en~tivlty of
detection which can be achieved with nucleic acid
: hybrid~zatlon i3 the rate of hybridlzatlon which can
be atealned. The co~bination of tetection of R-RNA
and the u~ of the rate accelerating incubation conditions
described abo~e ~ake ~ e pos~ible to attain extremely
high senqltivity of detection of baceeria and other
organi~m~ in a very short period of time with the use
of ~ery ~mall amounts o sa~ple and probe. An illustrative
example of thl~ is de~cribed laeer.
To the bes~ of ~y knowledge there i3 no pr~or art
concerning the use of rate-accelerat~ng ~y~tems with in
olut~on hybridiza~ion ~est~ for deter~lning the presence
or abRence o a~ organi~ or group of organisms by
detecting th~ R-RN~, transfer ~NA, other RNA or DNA of
the organism~ of intere~t. Ther~ i~ also no prior art
of which I a~ aware concern~ng th~ u~e of a com~ination
o a rate-accelerating ~yqtem and the enzy~e-detergent-
sa~lR-probe ~ixtures to determine the pre~e~ce or
absence of a ~pecif~c organis~ or ~iru~ or group of
; 2n organism~ or vlru~ by detectlng th~ R-RNA, tran3fer
RMA, or other RNA or DNA of the specific organism or
~ group of organi8~ of iatere~t.
- 5t~p 3. A-~s~ying th~ Incubatior~ M~xture for
The signal that the 3a~p1~ contain~ the target R-RNA
lecules ~and thcre~ore th~ target organi~m)`i~ the
: pre~ence of hyb~d~zed prob~ in th~ i~cubatlon mixtur~.
Thus the incubatlor~ ~ixtur~ r~t be asa~yed for th~ presence
,
:
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987
-3~
of hybridizPd pr~be at the end of the incubatlon period.
It is desirable tha~ such an assay be ea y to perform
and rapid. For this a~say the lncubatlon ~ix i~ pro-
cessed by utilizing hydroxyapatlt~ (HA). Under the
5 proper condition~ HA binds R-RNA rapidLy and oompletely
but doe~ not bind ~he non-hybridized probe molecule~.
If a probe molecule is hybridized to a target R-RNA
molecule the probe also bind~ to the HA because tt is
phyqically attached to the R-RNA.
Detection of organi ms by detecting their R~
ls a feature of the invention. The abllity of the HA
to bind R~ or R~A ln general, in econd~, whlle no~
binding ~ehe probe at all, ha~ allo~7ed the developDIent
of a hybrldization as~ay method whieh take~ nute~ to
perfor~a, has grea~ flexib~lity and whieh adapt~ well
for handling multlple ~amples. I~ addition the sample-
detergent-enzgme-probe incubation mlxture, can be diluted
~n~o the appropr~ate bufer and directlg proce~sed to
as~ay or the pre~enc:e of hybridized probe.
HA i~ known in th~ art a~ a ~ubstance used for
as~aying hybridiza~ion of probe The as~ay T~lethod
d~scribed h~e" whlch ha~ great advantag~ oYer ehe
prior art use~ of EA (Brenner et al., Analytical 3iochm
(1969 ( 28 p . 477), can be carried out at roo~ temperature
a~nd w~ll work o~rer a temperature rang~ of about 15a C
to abou~ 90~ C. It ha~ fewer ~tep~ as~d doe~ no~ require
heating at each s::enerifugatior~ ~tep; it casl be carried
ou~ ln th~ pre~enc:e or ab~ence of de~erg~n~c~ such a3
Zwltterger~t tCalb~;oche~, Dan Diego, Cali. ) ant sodiu
lausyl 3ulfa~ce. I~ i~ 3 - 5 tim~3 fa~er, aIld a ~ingl~
,,
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1 ~ 78 9~7
-39-
assay can be done in 3 - 5- minuce~. It requires about
5 time~ leqs HA, Deter8~nt concentration can range
from O to lOZ, wh~le ~he pho~phate concen~rationC can
range from 0.1 M to O.2 M depending on the type of
assay. The method can also be readily adapted for
handling multiple sample3.
Methods o~her than HA are avail ble to assay for
hybridization of the probe. The3e include enzyme assays
such a~ the Sl enzyme method, ~ize separation methodq,
and a variety of sample immobilization method3. The
probe.~ discus~ed here can be used effectively with
these ant any oeher method of conducing hybridization
ant hybridization a~ ay~.
Procedures for th~ Product~on
~
~: Different approsche~ can be used to produce group
spec~fic prob~s. All of these approaches but one, rely
on differen~ial nucleic acid hybridization methot~ to
ide~tify and purlfy the group speciic probe sequence~
2G Procedure A:
The ~08t u~ful procedure for producing group
speclfic R-R~A probss uses recombinan~ DNA methodology.
~ ~ ~he ~ep~ in~olved in thi~ procedure follow: (The
::~ pecific detail~ of ~tandard DNA recomblnant techniqu~s
; 25 ar~ de~cr~bed i~ ehe book,
Manual, ~. Maniati~ e~ al., Cold Spri~g Harbor Publication
(1982))
olat~ n~cleic acid fro~ a ~peclfic organis~
:: of in~er~st. Standard i301ation ~ethod~ are
; ~ 30 u~ed.
,
.~:
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~l~'789
-40-
2. IJsing t~iQ i~olated DNA, clone the R~ A
~enes of thi~. organi m and then produce
arge a~o~nts of the r~bosomal gerle DN~,
u.~ing ~tar~dard DNA recomblnant tec}~ology,
a3 qhow~ in Man~ ati-~ et_al ., ~upra .
3. ~educe the ~-RNA gene DNA to shor~ pieces
with re~tr~ ction enzyme~ and make a library
of tl~ese ~hor DNA pieces, u~ing ~tandard
DNA recombinant methods, as shown in
ManlatiY et al., supra.
4. Screen the library and ideneify a clon~
whieh contain-~ a short R-RNA gene sequence
which hybritlzes only to R-RNA fro~ other
Dlember~ of ~he taxonomic SPec~e3 of the
organl~ of lnt~res~. Isolate e~hi~ clone.
It con~s~n3 a Specie3 9peclfic DNA ~2quence
whlch i~ eomple~ntary only to ehe R RNA of
the speolfic Specleq to wh~ch the organism~
of interest belongs.
Screen the library further a~d identify and
i~olate th@ following clones: a) a clone
which contain~ a DN~ ~equence co~ple~sentar~r
to R-RN~ which will only hyb~id~ze to R-RNA
froDl ~emb2rs of the taxono~ic Genus to which
S ~he organi~D~ of in~e~e~t belon~i) a clone
which contain~ a DN~ ~equenc~ complementary
to R-RN~ which will only hybridlz~ to R-RNA
fro~ ~e~be:c~ of ~he taxono~ic Ort~r eo which
: ~he org~is~ of lntere~t beIong~; c~ a clone
: whlch coatain~ a i)NA sequence co~plementary
eo R-}UaA w~ich will hybridize only to R-~tA
: ~ fro~ ~eD~b~r~ of th2 taxono~ic F~ily l o which
the organi~ of~ in~ere8~ belon~ a
clone whic~ con~ain~ a !)N~ ~equenc~: complementary
to R-RNA wh~ch will hybridize only to R~ from
me~ber8 of the taxonomic: Cla~8 to :which ~e
organi3~ of intereslt belong~~p and e) a clone
whic~ conta~nY a DNA sequence co~plesl~entary:
~o ~-}IPaA'whlch w~ll hybridize eo R-RNA ~ro~
: 40 ~ : a~ man~jr diferent 1~fe forTIl~ a~ po~ibl~.
:
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1.~7~387
- 4 1 -
The foregoing clone qelectlon ~che~e i~ only
one of a nu~ber of pos~ible ones.
Standard method~ of cloning and 3creening are
to be utilized, a~ discussed in ~aniati~ et al.,
supra .
5 . a) Produce larg~ amount~ of each clone ' s DNA.
From the DNA of each individual clon~ isolate
asld purify only the DNA sequence which is
complementary ~o R-RNA, uqing one of the many
method-~ existing ~co accomplish thi~ , e O g., as
in Maniatl3 et al. ~ .~upra.
b) In cer~ain instances the total DNA present
in ~ clone is useful as a probe ~ ~n which ca~e
the total DN~ i~olated from ~he clonlng vector
~s uced.
c) In certain other in eance~, the DNA single
strant of the cloning vec~cor which contain~ the
DNl~ ~equ~nee comple~nentary to R-RNA i~ used as
a probe. In such case thi~ ~trand muse be
isolated and puxifled, using on~ o:E ehe various
method3 which ex~ ~t to aceo~plish thi~, a~
de~cr~bllad by Maniatis et al.
6. The probe DNA obtai~ed in 5a, 51:~, and 5c must
. be rnar~d in ~ome way ~o tha~ ie can be
` ~ identifi~d irL the a.~say mixrur~. Many different
ki.nts of T~arker~ can be uq~d, the ~o~t frequently
u~ed ~rkder bein8 radioactivity. C)thers include
fluoress:ence, enzy~es, and biotln. Standard
: methoâ~ are uqe~ for marking the DNA, a~ set
oue in Mania~i8 et alO, ~upra.
7. The ~roup 3pecific R-R~A gene ~equenc~ in the
clon~ng v~ctor exi~ts in a double strand ~at~.
On~ of th~e 3trand~ 1~ complementary to R-RNA
and w~11 hybr~dize wlth it. The o~her strand
wiIl not hybridize to R-RNA but can ~e used to
:: ~ produc~. ~arked group specif~c ~quence~ com~
mentar~ R-RNA. Thi3 i don~ b~r u~cilizing a
D~A or RNA pol~era~e and nucleic acid precursor
. ~ :
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~ ~ .
::
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.
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~.~ 7~38
-42-
molecules whlch are marked~ The enzyme will
utilize t~e marked precursor~ for ~ynthesizin~
DNA or RN~ u~ing the DNA ~trand as a tem~late.
Th~ newly ~ynthes~zed mærked molecule will be
comple~entary to R-RN~ and can be used as a
group 3pecific probe. The template DNA can be
re~oved by various established means leaving
only single ~trand ~arked nucleic acid, as
described in Mania~i~, et al., ~upra, and the
artlcle by Taylor et al., in 810chemica and
Biophy3. Acta (197~ 2. p. 324.
Procedure B~
: Several enzy~es can utilize R-RN~ from any ~ource as
a te~plate for ~h~ ~ynthesizing of marked DNA complementary
to the entire R-RNA sequence. Group qpeci~ic sequences
co~ple~entaxy only to the R-RNA of a particular c~as3 of
-~ organis~ can b~ i~olated by a hybridization ~election
proc~s~. The fraction of the synthe~iz~d marked DNA
~ : which hybridize3 onl~ to the R~RNA from member~ of a
:~ 20 specific class of organi3m~ ca~ be i~olated by standard
;~ hybridization proceduse~. An example of ~his proce~ i3
pre~ented her~inafter. Such a probe can be produced in
uficiene qua~title~ to clone ~ i5 de-~cribed in A.
The~ base sequence of ~hi~ clone can be determined by
tandard met~od~ and the ~eque~ce u~e~ to direct the
produc~ion of the probe by chem~cal synth~is using
ea~dard method~.
: Procedur~ C
30 : Th~nucl~otide Qe~uences o~ R-RNA fro~ wid~ly
di~ferent orga~is~.have been~d~teræined. Çroup specific
e~uences ~ilar to a specific:group of or~anis~ can
., ~ :
~,
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~1 ~ 789~37
-43
be identified by comparing the~e known sequences. A
sequence complementary to thi~ group speclflc R-RNA
sequence can then be chemicall~ synthe~ized ant marked,
using standard methodology.
Psotuctior~ of Specific Probes
Complementary to t-RNA
While differenc approaches can be used to p,roduce
specific t-'RNA probe3, the same ba~ic approache3
de3cribed for producing R-RNA probe~ can b~ used to
produce t-RNA probe~. Standard ~ethod~ are available
to i~olate indi~ridudl t-RNA species and gene~ and ~hese
are well known in the ar~. The for~ of ~he probe may
be DNA or RNA, and the l~ngth of the probe may be 12 to
:~ thousands of bases long. The probe need no~ be perfectly
cosnplemerltary to the nucleic acid it i~ ~pec:ific for >
i.e., the targe~c nuole~c acid, and the whole length of
the probe need not be comple~entary to the tar~et molecule.
: ~ .
`; Productlon of Spec~f~c. Probe~ Compîemen~a~y
The sa~e ba ic approache3 used to produce ~pecific
probe~ co~Dplem~ ary to R-RNA can be used to produce
; i ~ 3p0Cii~iC probe~ for ~pecific elasses or populations ~f
mRNA, hn~A, ~nRNA, or psRNA. The me~hod~ for isolating
each C:12~!~ of RNA and ur~her frac~c~ onating it are well
~S ~ known in the art . Again the form: of the probe ulay be
- DN~ or~RNA, and the leng~ch of the probe ~ay ~a~y from
about 12 to thou~ands of bas~s long.. The co~sple~entary
region of the probè need not be perfectly complem~ntary
eo the targe~c nucleic~ acid and the whole lengt~ of the
30 probe n2ed not be compl2mentary to the target moleeule.
~ ~ .
~::
.
789~7
- 4 4 -
Isolatin~ Sample Nuclelc Acid
Standard prior art me~hod~ can be uqet tO i301ate
nucleic ac~d from the ~ample~ to be assayed. One
standard method of nucleic acid i~olation and purification
is pre~ented in the example section and iQ alss discu.~sed.
in Maniata~ et al., supra.
A new ~echnique for making nucleic acid3 available
for in ~olution hybrldization without performing
purificat~ on step i~ described hereinafter.
Perfonnin~ the Nucleic Acid Hybridization
An approprlate amount of marked probe ~9 mixet wi~h
the sample nuc:leic acid. This mixture i~ then ad~ustet
to a specific ~ concentra~ion (NaCl is u~ually used)
~ and ths entir~ mix incubated at a ~pecifi~ temperature
:~ 15 for a -~pecific ti~e period. At ~he ~nd of the ti~e
per~ od the mix~ure i~ analyzed by perfonaing a hybridi-
zation as3ay. Many different combination~ of salt, solven~,
~ucelic ac~d conceneration~, volum~ , and temperatures
; exi~t which all~w nucle~c acit hybr~dization. The pre-
; 20 ferred c~binatio~ depenting on the circum~ta~Pes of
~ the a~say. It i~ important, however, ~ha~ the criterion
-~ (s~e "Defin~tion~) of ~he hybridization ~tep~ be idsntical
~o criterla used to iden~ify and select th~ group probe.
If the criteria of the hybridization step is different,
the probe speciiclty ~ay change. See: "Repeated
Seque~e~ in DNA",.by Britten ant Ro~n~, Sc~ence (1968~
161 p. 529; "Kinetic~ of Renatura~ion of DNA", by Wetmur
and Da~it~on, J. M~: Biol. (1968) 31 p. 349; "Hydro-
x~apatit~ Technique~ for Nucleic Acid Reas~ociaeion", by
Rohne and Britten~ Proced~r~ in Nucleic Acit Research
. :
,~
I.
,'` ''''
. :
.':
'1,'4~
.'
:
~L~7~98?7
-45-
(1971~, ed~ . Cantoni and Davie~, Harper and Row, Vol 2 ,
p. 500.
Two different approaches are used with regard to
the amount of probe and ~ le nucleic acid presene in
the hybridization mixture. In one, the exces probe
m~thod, there is ~ore probe present ~han sample nucleic
acid, in thi~ ca~e RNA. With the other, the excess RNA
method, there is more R-RNA present than probe. The excess
probe method i3 ehe method of choice for detectin~ the
presence of RNA ln unknown samples. I~ ha3 se~r~ral
advantageq which are discuc~ed below. See Table~ 1
and 2 for further discusslon of theYe ewo approaches.
Using the exces3 probe method9 the d~tect~on and
quantita~lon can b~ done with just one lab a~say point,
if the proper RNA prone i8 available. If the hybrit~-
za~iorl has gone to completion the a~nount of probe which
ha~ hybrldized i~ a direct ~ea~ur~ of the amount of
R~A preYent in th~ sample. The fact that the probe
hybr~dize~ at all indicateQ ~hat RNA 1~ presen~, and
-~ 20 the amount ~of probe which hybridiz~-~ indicates . he a~nount
o~ RNA p~esent ln the sample;
Making 311r~ eh~t the hybridization ha~ gone to
completlorl in a known l:lme i~ i~nportant in order to
quaneitate thg RNA. l~ read~ly dorle by adding
2S enough probe o ~nsure that tha hybri~izatiorl goe~ eo
co~pletion in a ~elec~ced ~ime period. The more probe
: addedt the ~a~ter eompletion is rearhed. Thlls the excesq
probe me~chod provite~ a mearls to en3ure ~ha~ the hybrid~-
: za~on ha~ gon@ t8~comple~10n and to know whesl thi3 ha~
occurred.
~: :
: :
-
:
-4~-
In contra t, the deteotion and quantitatlon of RNA
can'~: be done wlth,one lab as~ay point when using the
exce~s R-RNA ma~hod. In addition, the ~ime when the
test poin~ ~hould be taken cannot be predicced in the
excess RNA me~hod. Unknown samples with small amount-~
of RNA w~ll hybrldize muoh more slowly than sample~
w~th large amo-mt~ of RNA.
The As~ay for H~rLdizat~on
The ~ignal that RNA of the specific group ~S in the
.~ample 1~ the preqence of double str~nd market prob~.
Man~ different method~, well document~d iFI the literature,
ax~ available for a~sayislg the hybrid~zation mix~ure for
the presence of marked probe in the double 3trand for~.
The choice of me~hot depend~ upon th~ me~hod chosen or
th~ hyb~idization ~tep, the eompo~ition of the hybrlti-
zation mixtur~, th~ type of marker on th~ probe and other
factors. One co~monly u-ced method i~ descrlbed hereinafter.
Se~ also Wetmue and D~vit~or~, Kohn~ and Britte~, and
l~ et al., ~upra. Al~t) the ar~icle by Fla~ll et al.,
Eur. J. BiochR~. (1974) 47 p. 535. And al~a, the article
by Maxw~ll c~ al., ~ leic Acid~ Re~a~ch ~1978) 5 p. 2033.
I21 all ca~ owe~ex ~ protant to either
as ay at or abo~7e the 8am~ criterion u~d for ~he hybrldi-
za~ion reactio~ or at a criterlon at which hybridlzation
canno~c occur.
Quan~itatlon o~ Nuc:leic Acid Sequence~
by ~ucleic Acit HYbr~d~:zation
Th@ ~uanti~cy ~f nucleic acid pre~en~ in a ~ample can
~` be det~rmlnQd in ~everal ~;3y~ by nucleic acid hybritl-
30 zation, u~ing method~ w211 known to . hQ art. The two
.
,
:
.
78~87
- 4 7 -
me~hod~ are di~closed hereinafter u~ing the example
of quant i ~ at ing R-.RNA .
It will be unterstood that th~ pre~n~ ;nethod i~
generally appllcable in any ca~e where ~ t is neoessary
5 to deter~ne the presenc~ or absence of organism~
which contairl RNA or DNA and that ~uch inelude~ bio-
logical sarnple~ such a~ sputum, seru~ tl~ue swabs,
and other ani~al fludis ant tissues as well as industrial
and pharmaceutleal samples and w~er. Specific tetail~
10 of the approach will charge depending orl wh~th~r R~IA
or DNA is being quantitaeed but the general approach
i~ th~ -~ame for both DNA and RNA.
;
, i,~
i~
.
~ 7~9
-48-
TABLE 1
-
EXCESS SELECTED PROBE METHOD
PROBE: The probe is a specific, selected, marked
sequence from a member of bacteria group B,
which represen~s 10 percent of ehe bas~
sequence of the R-RNA, and hybridizes
completely with R-RNA from group B bacteria,
but doe~ not hybridize with R-RNA from other
: organism The probe cannoe hybridize with
itself.
A. Positive Hybridize to a) One percent o
Homologou~ completion the probe wlll
Control and assay for~ touble
. for double strand mole-
- ~ 0.1 mlcro^ s~rand pro~e cule~.
- 15 gram Probe
b) Thi~ is a direct
measure of ~he
~0 3 R-RNA sample.
m~crogra~$ The n~mber of
: 20 Sam~le probe molecules
~:: group B hybridized equals
R-RNA the number of
:~: . R-R~A ~olecule
: present.
:: 25 ~ B. H~tero- Hybridize to: The~ probe does
l~gous completion not hybridize
Control: and assay with any R-RNA
for double but R-RNA from
û.~l mlc~o- strand probe group B bac~ria
gram~ Probe
30 ~
10 3 micro-
gram~ Sa~ple
human R-RNA
.
,, ::: ::
? ~ :
, ~ ::
,i,: ~ :
'~ ~
':
~'
.
':
:: :
',.`
::~
`:` ~ :
,:~ .
, ........................................................... .
... .
.
~L~789~37
- 4 9 -
TABLE; 1 ( Cont ' d)
C. Unknown Hybritize to a) If no group B
Sample completion R-RNA is pre-
n 1 m~ c~o and assay sen~, no probe
grams Probe f~r ~Ubleb will hybridize.
b ) If group B
+ R-RNA is pre-
Unknown will hybridize
Sample and form double
s tr and mo 1 e -
cul~s.
c ~ The number of
probe molecules
hybrldized
equals the
number of group
B R-RNA mole-
cules present
in the sample.
d) If one pereent
of the probe
hybridi~e-~ ~
group B R- RNA
is pre sent
~:: since ehe probe
. wa~ selected so
: that it would
hybridize only
~; wi~ch R~RNA fro~
~ ~ ~ a group B
-~ ~ 30 : bac~eria. Since
the probe will
only hybridize
to group B
R-E~NA, ~he
pr~sence of
: other R-RNAs
: : will not inter-
.~; ~i ~h ~ne
~ction or the
quan~citation of.
any bacterial
R-RNA present .
:`: ~::
.` ::
,. :
.
.
,~ -
398~ -~
-50-
TABLE 1 (Cont'd)
e) Usin~ a selected probe
nake~ it easier ~o ensure
~hat the hybridization
is complete. A
selected probe repre-
sen~ing 10 percent of
the R-RNA se~uence will
hybridize 10 times
faster ~han a probe
which is representa-
eive of the ~otal R-RNA
sequence.
f) The detection of R-RNA
in ~ner 1 is not
poscible ~ince the
probe hyb~idizes only
with group B R-RNA.
The ~en~itivity of
de~ection o group B
R-R~A i~:exeremely
: ~ high.
:
D.
T~e exces probe me~hod need~
jus~ one as~ay point~ orter
25: t o de~ect and quantiea~e group
B organi~m~.
:: :
: : :
::
:
,
~ æ
...
.
~789~17
-51-
TAB~E 2
EXCES5 R RNA METHOD: THE USE OF A SELECTED PROBE
PROBE: The probe is specific, selected, marked
sequence from group B baceeria, which
represeneq one-centh of the R-RNA base
sequence of one member o group B. Th~
probe hybridize co~pleeely with R-RNA
from group B, but does not hybridize to
R-RNA from other organism~. The prob~
canno~ hybridize with it elf.
A . Posit~ ve Hybrid~ze to a) The fraction
Ho~ologou~ completion of probe which
atld a3~aY a direct
Sa~ stra~d prob~ mea~ure of ~he
gra~ ~roup R-RNA a Id ~his
+ ca~@ 10û per-
cent of the
10 3 ~nicro- probe can
gram~ Probe hybridize.
b3 Thls percene-
`~: age is r~oe a
`~ ~ : measuse of the
a~olmt of R-RNA
;~ ~ ` 25 pre~ent. In
order to deter-
mine chis ehe
kinetic~ of
th~ reaction
. ~ t be deter-
~ined.
.
.~ ~
,.
;
.~ ,,:
~78~387
-52-
8. Hetero- Hybridize to The probe does
logous completion not hybridize
Con~rol and assay to non-bacterial
for touble R-RNAR.
0 .1 m;cro- s trand probe .
grams human
R- RNA
:: +
Probe
10-3
micro-
gr ams
C. Unknown
Samole Hybridi~e to a~ If no group B
completion R-RNA is present
Samole and a~ay i~ ch~ ~ample
for double ehere will be
serand no hybridized
probz. probe.
P ~ obe
;: . b) If group B
10 3 R-R~IA i present
micro- the probe will
~ grams be hybridized.
:: 25 c) The amoun~ of
R-RNA can ' t be
determi~et from
ehe perceneage
: hybridization at
30 . the comple~ion o~
~he reaction . In
order to de termine
this the kinetics
of the hybr idiza-
: 35 tion ~ust be deter-
minet. Since the
pro~e will hybri-
dize with only one
type o~ R-RNA, the
: 35 kin~ic determina-
tion i~ simple.
:
'~
~:~
'b~
.~
:
~78987
~ 5 3 -
TABLE 2 (Con~: ' d~
d) If 100 percent of th~
probe has hybridized
with the sample, this
meaII~ that group B
S R-RNA is presen~ in
the sample. It does
not indicate that only
this R-RNA is prPsen~.
Other R-RNAs which do
not hybridize with the
probe may also be pre-
;: sen~c in ~he samp le .
e~ ~ 100 percent of the
probe hybridizes with
lS the sa~pIe, it is
pos~ble to :~pecifi
cally quantieate the
group B R-RNA in the
present of human R-RNA
by de termining the
l~in~tics of hybridiza-
tion :o~E the probe with
the sampLe R-RNAv Sinc~
he prob~ will: hybridi~e
o~ly ' w~th group B: R-R~
uch a kineti~ reaction:
will have only one
coE~Ipon~nt, the qne from
reacting with group B
; 25 : ~ R-RNA.
f) There are ~ituations
:: : : wh2~e eh~ hy~ridiza~
tlon Gan ~ t 8 to com-
pl~tion. In thi~ me~h~d
30- ~ . the~sample R-~NA musc
drive: he hybridization
to :cc~ eion, since
only a very smalL~ :a~unt
:of probe i~ pre~es e.
: 35~ If :th~re ~ no~ su~fi-:
cie~at~ R-RNA in the
sa3~ple:, the hybr~diza- -
t~on ~ill xlo~ be
:: : : : compleeod. The inte
3S: : ~ pretation of such a:
ltuation i~ d~3cus~ed
: bslow.
~8~3~37
- 5 4 -
: If hybridization of unknown
s~mple results in 20 percent
hybridization of the probe
- at the usual assay time,
S it is not possible to tell
if th2 reaetlon is complete
with only one time poine.
It is neceqsary eo take
another poine at double
~h~ original ti~e to
deter~ine if ehe hybridiza-
tion value increases. If
ic does not iner2ase then
: the hybridization is complete.
: 15 In thi~ ca~ the R~RNA is at
such low concentration in
the ga~pl~ that the proba is
in exee~, and the nu~bex of
R-RNA molecules present in
: 20 the sample i~ equal to the
:~ ~umber o~probe ~olecule.q
~ hybridize~.
: If the hybridizaeion value i5
~:~ : increa3ed, the hybridizatIon :
`:: 25 wa~ not over a~ ~he first
ime-point. A third time-
: poin~ mu9~ ehen be done to
. : tetermine whether the
r~action wa~ over at the
~ second time point.
: The ~xce~ a~ple R-RNA method
need~ T~ultiple as~ay point~ :
in ord~ o deeec~ and ~ :
:~ 35 : quan~i tate~ and i~ much
more ~isne-con~u~ing :~ ~hat
: th~ exce~- probe method. ~ :
.
,., .
: ~ ~
.. ~,: : :
::
; ~
~ , ::
.
.
-55-
USE OF SELECTED PROBE5 COMPLF-ME:NIARY TO ONLY A
PARTICULAR E~CTION OF THE R-RNA SEQUI:NCE FR0~2
A PARTICUI,AR SOIJROE TO DETECT R-RNA VERSUS USE OF
IJNSE~GTED PROBES Cû~?l~:NTARY TO THE ENTIRE R-RNA
SEQUENCE FROM A PARTICULAR SOURCE TO DETECT R-RNA
On~ aspec~ of my invent~on, which comprise~ using
specifically selected probe~ comple~entary to only a
particular fractior~ of the R-RNA sequence~ to d~tecc,
quantita~e, and identify R-RNA ha~ ~mpor~cant capabilities
10 and advantages over anoth~r a~pec~ oi~ the invention, that
of u~lng unseleceet probe~ or 3equences co~plementary
to the entire R~ equence to d~tect R-R~A. The
advantage~ of u~inE5 a 3elected probe in b~Eh exce~
R-RP7A and exc~s probe hybridization methodologie3 are
~ 15 sst orth below. The proble~s with uslng a co~pletel~
repre~en~cative probe are al~o pre~nted.
Th~ advan~czg2~ of using a selected probe ov~r u~ing
a cor~plQt~ly repre~neative R-RNA prob~, with exce~s probe
hybridization, a~ well as wi~h exce~ R-RNA hybrldization,
20 l~ set out below:
Proble~ wit~h CaDpl~ely A~ of Us~ng
1, R~ ected in a ~e selec~ed probe ~ be used
sa~le ~rith ~he ~:ess pr*~ ecr~ d specifically
detPr:~ ~ ~ of l~ ence of a pa~iculæ R-RNA,
prese~lt. T~3 ~ probe ~n't ~n an ~ ~a~ple wh~ used
'be u~ed to spec~ y detect i~ an exce~s p~robe }ybridizati~n
~nd qu~ntitaee 1~ p~e of D~t}d. Tnls ~ be dc~e with
. ~lie., w~ hs exce~ pro~a prese~e of R~R~ frasl o~r
~g8~.
,
.
,~,
,
.~ .
~,7~3~3~7
-56-
2. As s~ated abave, t~ exces~ The use of a selected probe
Dro~e ~thod carrlot be used with n~ke~ it pocsible tl~ use the
~s probe to detect or quanti- excess probe m~t~od for
tate the presence of a particular detect~n8 and qu~ticating
R-~ in a s~ple. For this pur- the presenf e o~ a pæ~i-
pose the probe ~st be used ~n cular R-RNA in art ~awn
the excess R-RNA m.ethod. sa~le. This greatly
s~li fies the task.
The excess R-R~ nE!thod i~ ~ch
more t~ne cons~i~ng, re~es
nuch more ~ric, and is m~ch
D~re colT~licated ~ the excess
probe n~thod.
Advanta~es_o tlle Exce~s R-RNA Hybridization Method
Probl~ with ~pletely Adv~tage~ of Us~
lle~re~ Rde Seleceed Probe
1. R-RNA cal ~ deeected in The seiected pro~e c~ be used
al ur~awn sample with this to spec;fically tetect and
: probe, but in m~r cases there quantitate the presence of
i no way of ~etermining thE~ a pæticular R-~NA in æ
~:ype or quanei~y of R-~ ur~ow~l sa~le ~n all
which is presen~. l~s in si~ation~. This c~n be
m~ instances the pro~e . dar2e even in the presence
nnot be.used to specific- of l~g~ a~unt~ of R-RNA
ally detect and qu~titat~ from other organisms.
ehe presence of a p~ti~ulæ
R-~ i~ an ur~ sa~l~.
2. In mar~ ca~e~ ~ sensi- With the selected probe the
~: ti~ r of detec~o~ of a pre~nce of R-R~ fra~ oth~r
~: 30 spec~ mLtet organism~ d~es not la~ar the
by the pres~æe o E~-~A sen~itivi~y of detection of
frc~ oth~r o~ga~s~. a particular R-~A.
3. In m~ case~ ~ere it is The dbtection e~ld quantitation
. possible to det~:t and ~ti- of the presence of a p~ticular
~te the presence of p~rticul~ R-RNA i~ u~xh easier when a
R-RNA9 lt require8 a lo~ of selected probe is utilized.
. ~ ~.
., .
, .
., .
,''
'~
,;
".
. .
... .
-57-
Il lu~ trat ive Embodlment _
My invention,. illu3tra~vely, m~y be u~ed to
determine whether a ~ample contain~ any member of a
partlcular gs-oup of li~ring organi9m~. Th~ m~thod,
5 de~cribed ln the following example~ a te~t which
may be u~ed eo detect and quantiaee th~ presence of a~y
member or member~ of a partlcular group of baceeria in
a sample, even in the pre~enc~ of large n~ber o~.
organ~sm~ which are not mellDber~ of thae par~ cular grsup .
A~ se~ forth in the examples, applicant's method
irnolve~ f~r~t producing a group 3peciflc R-RNA probe
wh~ ch, at a pecific criter~o~, hybridlze~ eo R-RNA
fro~ any me~ber o the ~peclfic group o~ interest, but
toe no~ hybridize to R-RN~ fro~ a¢ly ot~er organims.
15 The u~e of ~uch a probe in a nuclelc acid hybridiza~ion
te~e allows che de~ction of any n~ember oi~ that qpeoifiG
group, ~ven in the pre~ence of large number~ o other
orgarlism~ . .
ExaD~ple~ of the practic:e of the in~ntio~ are lis;ed
20 la~cer. Each exa~ple involve~ the product~on o~ a marked
nucleic: ac~d pro~e .which will hy~rid~z~ only ~ieh R-RNA
roE~ ~e~er~ of a partlcular group of organi~Ds.
The ba~lc ou~l:llne of the ~ethod used ~o produce
each pro~¢ ~ ollow~:
2S 1. Producc ~a~ d nucleic acld co~ entary ~o
the R~ of a member of th~ group o~ inl:erese.
. Hybrit~ze thi~ D~ to R~RNA fro~ a ~e~ber o
the group o~ ~roups of :3rgan~3~ evolu~ionarily
~o~t clo~el~ related to thls group o organisms
fo= ~-h1ch th~ p~obe i~ to be sp-ci~ic, Selecc
~'
:
~:
'1;~78~387
-58-
the fraction of t~ marlced nuclelc acld which,
at a ~pec,ific criterion, to~ ot hybridize to
R-RN~ froD~ a ~mber of this clo~eat related
group o~ organi m~. This frac~lon i~ ~peci~ic
for the R-RNA of the organi~m gro~p of lnterest
and doe~ not hybr~dize wi~h R-RNA ~rom the most
closely related group or group~ or arly other
organis~.
Exampl~ l: Produc~ion of Probe Whlch Will Hybridize
~In ~ ~ypical ~tuation, abou~ lO~ -107 ~ammalia~
cells are grown in~a ei~3u2 cul~ure pla~ce at one tim~.
Bacterial ~pecie~, expecially member~ oi~ the Eaxonomic
Cla~ Mollîcute3, are known to conta~rlat~ ti~3ue culture
cell~. Memb~rs of the t:las~ ~ollLcue-J, ~llke 1~03t
~: ~ other bact~ra, are noe r~adily eli~ina~ed b~ arlt~biotic~,
and aré trouble~o~ conta~inant~ o~ c~ll culture~. Many
- diffe~ent ~olllcute~ specie~ have been deeeeted in ti~lSUe
c~lture cells:. If ~u3t one o the~org~l~m~ i~ pre~ent
: 20 in the cul~ur:~ pl~te9 it ha3 the poten~al~ ev~n in the
pre~en~e o~ ant~biotic~, ~o ~ul~iply a~d produce hundreds
o~ organls~ p@~ cell. Such organis~ are capable o~
erin~ th- act~Yl~ o~ cells, there~ ~ff~c~g the :
result~ of ~ariou~ s~udle~ and:the marke ablli~y of cell
25 ~ culture~product~.
Prio~ art~ethod~ for detecting eh~se or~ani~
involve:baa:ically:~ualitatlve te~ts, the E~t commonly~
u~ed~being growth ~e~t~, differe~ial ~aiain8 te~t~ and
i~Naolog~ a~ay~. The grow~h te~t~, while quiee
: ~:
~,:
1.~789~7
o59
sen~itive, ~ake 3 - 6 week~ O They have . he adtitional
disadvantage that many organis~ns are difficult or
i~pos ~ ible ~o grow.
While the actual detec~ion senqiti~rity of che
~taining meehod i not: known, it i~ known that more
than qeveral organisms per cell ha~e to be pre3ent.
Immunologic tes~ are quali~ative test~ ant in~rolve
using ane~body toward a particular specie While ehey
can be car~ied out rapidly, they are nc~t very sen~itive;
furthermore~ many different antibod~e~ would be required
to tetect all typ~ of Mollicutes.
The embodimen~ o~ applicant ' ~ me~ho~ described below,
i3 a test which may be u~ed ~o detec~ arld quan~itate ehe
presQnce of a~y. member of the group Qf jqlll bacteria,
includlng ~he taxonomlc Class Moll~cue~l, to detec~ the
presence of ~ollieutes in tis3ue culture, to detect the
presence of bact~ria ~n ti-~su~ which i~ nonnally free
of baeteria, a~d to detect ehe pre~ence o the bacteria
evea~ i~ the pr~ence o~ larg~ nber~ of ma2nrllal~ an ce11~ .
A~ ~et foreh in th~ example, applican~ ' ~ ~ethod
- in~olve~ fir3t maki~s~ a pecif~c R-R~A prob~ ~hich i~
complen~en~cary to R~ ro~ any bacteria but is not
eo~Dplem~ntary to ~alian cell R-RNA. The U~@ of such
a probe 1~ a rluclelc acid hybridization t~t allows th~
d~tection of any bacterla type, e~en in th~ pre3Pnce of
large numb~s of ~2am~lian cell~.
A detai}~d de~crlptio~ of thi~ e~bodi~en~ of the
in~ention follow8
~: :
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:
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~ ~ 78 9
-60-
Prepa~ation of R-RNA from Mammalian
and Bacterial Cell~ .
Mammalian cells are resu~pended i~ 0.3 M Nacl,
0.02 M Tri~, pH - 7.4. Sarko3yl ~ B adted to a final
concentrae~on of 1 percent to ly~e th~ cell3. Immediat~lv
up~n lysis an equal volume of a 1/l mLXtUre of phenol/
chloroform iq added and the re~ulting mixture shaken
vigorously for 2 minute~. The mixture i3 then centri-
fuged (8000 x g for 10 minute3) to ~eparate the aqueous
and organic phases. The aqueou~ pha~e i~ recovered, and
to thi~ is added another ~rolu~ of phenol/chloroform.
After shaking and cerltrifugaeion a~ abo~ve 9 the aqueou3
phase ~ g again recovered. To thi i~ added 2 volumec
of 95Z ethanol and thi~ ure i~ placed at -20 C for
2 hours to facilieate precipitatian of the ~ucle~c acid~.
Then the ~ixtur~ i~ centrifuged ~8000 x g, lO minutes)
in order to sed~ent the pr~cipitate to the bottom of
the tube. The liquid i3 ~hen removed. The p~lleted
nucle~c acid i9 redi~301~d in water. Th~e solution is
then mate to 0.2 ~ NaCl, 5 x 10 3 ~ MgCl2, 5 x 10 3
M CaC12. 0.02 M Tris (pH ~ 7.4), 50 ~icrograms per ml
of deoxyrib~nuclea~e I and incubated at 37 C for 1 hour.
Then add an equal volu~ of phenol/chloroform and shake
as abo~e. Cenerifuge a~ abo~e and recover th~ aqueouY
~5 pha~e. Ethanol preclpat the RNA a~ above. Centrifu~e
the pre~pitate a~ abo~e and red~_~olve the pelleted RNA
;~ in water~ Make ~hi~ solut$on to 2 M LlCl and place ie
at 4 C for 10 - 20 hou~3 in order to facilieate ehe
precipitatio~ of t~-h~gh molecular wel~ht RN~. Then
~: 3~ cen~riguge thls ~olueion to collect the precipaee and
~;
~;
:
,
~.
æ
789~7
-61-
ret~-~solve the precipitate in water. This preparation
of RNA coneains9 gr~ater than 95% R-RNA.
Bacterial R-~NA i~ lsolated in a s~ilar ~anner wlth
ehe following exceptions. In ~hose cases wh~re deter-
gent alone doex not lyse th~ bacteria, other meanq are
employed. Thi~ usually involves pretreating the bacteria
with an enzyme (lysozyme3 to make them susceptible to
lysis by sarko~yl. After lysis of the bacteria t~e
isolation procedure i~ as described above.
Purifled R-RM~ is stored at -70 C.
Produ~eion of Radioacti~e DNA Complementary
(3H-cDNA) to Mollicutes R RNA
R-RNA from the specie~ MYC~e1aSma ho~ini (M. hominis),
a member of the taxono~c class ~ , i9 used as
a te~plate to ~ynthe3ize radioac~e cDNA oomplementary
to M. homini R-RNA.
Thl~ cDN~ i~ produced by utllizing the abili~y of
an enzyme, reverse transcriptase, to utilize R-RNA a~ a
~emplat~ and produce 3H-cDNA com~lementary (cDN~) eo
R-RN~. Th~ r~erse tran ~ a~e reacelo~ ~i~tu~e
contains ehe foll~wln ~ 59 ~ Tri~ CL (pH - 8~3) 9
8 ~M MgC12, 0.4 ~M d~thiothreitol, 50 ~ KCL, 0.1 ~M
~I-deO:~y~chy~idln~tr~phO~pha~e (50 C~rie~ Per Dlilli~Ole~,
O . 2 ~M deOXYadenO8inetriPhO-~Pha~e, 0 . 2 DM deOXYOYtidi-
netr1PhO3Phate~ 0 . 2 m~I deOXY8UarlO~inetriPhOSPhate, 200
m~cro8ram~ per ~1 o~ oligodeoxyribonucleo~ide primer
~ate from E. coli DNA, 50 mlcrogra~ per ml of M. hominl~
R-RNA and 50 unlt~ per ml of AMN reverse transciptase.
'~
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-62- ~78~
This m~eure i~ incubated at 40 C for 30 mizlut~s .
Then ethylene dia~ine tetraacetic acit ~EDTA) (pH D 7 . 3),
~odiur~ dodecyl ~ulfa~ tSDS), NaCl and glycoge~ are
added to final concentration3 of lû 2M, 1 percent, 0 . 3
5 M, and 100 microg~ per ml re~peGtively. ~he solueion
i~ then ~aixed with 1 volume o~ phens~l~chlcrofon~ tl~l)
and shakesl vigorou-~ly for 2 minute~, ehen een~crifuged
(8000 x g for 10 minut~) and the aqueous pha~e recovered.
The nueleir acid~ a~e precipitatet by th~ addition of
2 . 5 volume of 952 e~hanol . Th~ precipica~ce i~ re~overed
by centrifugatiorl and redi~olved in ~ 901ution
ontain~ the te~plat~ R-RN~ and eh~ newly synthe.Qized
H- cD~A .
Thi~ solutlon is th~n"nakQ to 0.3 M NaOH and
incubat~d a~ 50 C for 45 r~l~nuEs~, and cool~d in ice
and neu~rali~ed with 0.3 ~f Ht:l. Two and on~-half ~olumes
o~ 95Z EtOH ara ~n added to precipi~at~ eh~ r~maining
nucl~ic acid an~ th~ re~ulti~g pr~cip~:at~ redissolYed
~n water. Thi~ ~olu~ion is ~he~ pa~-d over a S~phadex
G-10~ colum~ ~quili~tet to 0.3 M NaCl, 0.1 percent
carko~yl and th~ ~xclu~d volum~ r~covored. Thi~ ~nlu~ion
hanol pr2cip~t~t~d a~d th~ r~uleinE~ p~cip~ tate
red~.3~01~r~d i~ a $mall volu~ of ~at~ Th~ p~oc~
descri~ed in eh~ p~æagraph re~o~re3 eh~ ée~plaee R-RNA
ant an~ re~ainisl~ pr~c~or ~ater~al fross eh~ 3H~cDNA
pr~p~atioT~.
3~cDNA 1~ eh~a hyb~ z~d to IS. ho~lnl~ R-RNA
co ensura that le i~ lndeed co~ple~entary to ehl~ R-R~A.
Th~ hybridlzation DliX~ oo~ t~ o~, 0.0S mic~ograms
of ~ingle strand 3H-~DNA, 20 mic~o~r~ of 1( ho-lniJ
,.;:
:
. * Trade Mark
`''''
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7~9
- 6 3 -
R-RNA, and 0. 48 M PB (phosphate buffer) in 1 ml.
This mixture is incubated for O . 2 hours at 65 C
and is then diluted to O.14 M PB and pa~sed over a
hydroxyapatite (HA) column equilibraeed to 0.14 M PB
and 65~ C. 3H-cDNA.hybridized to R-RNA absorbs to the
hydroxyapatite (HA) column while non-hybridized
3H-cDNA passes through the column. The hybridized
3H-cDNA is then recovered by elution of the HA column
with 0.3 M PB. This frac~ion is then dialysed to remove
the PB, ethanol precipitated to concentrate the nucleic
acid, centrifuged and the nucleic acid redissolved in
water. This solution is then treated with NaOH as
described above in order to remove the R-RNA. After
neutraliza~ion, addition of glycogen carrier and csn-
centration by ethanol precipitation, the 3H-cDNA is
redissolved in a small volume o~ water. This solution
contains only H-cDNA which is complementary to M. hominis
R-RNA.
Selection of 3H-cDNA Which is Complemen~ary ~o
~; 20 M. hom nis RNA but is not Complementary to Human R-RNA
The purified H-cDNA is further frac~ionated by
~ hybridizing it with a great excess of human R-~NA. The
; ~ hybridiza~ion mixture consists of 0.05 micrograms o
;~ H-cDNAj and 40 micrograms of human R-RNA in one ml of
Q.48 M PB. This is incubated at 68 C for 1 hour and
~ the mixture is then diluted to 0.14 M P8 and passed over
: ~ HA equilibrated to 55 C and 0.14 M P8. The fraction
(about 50% of the ~otal) which does not adso~b to the
~;~ : HA (i.e., 3H-cDNA not hybritized to human R-RNA) is
colleceed. This fraction is repassed o~er a new HA
'
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-~4-
column under the same condition~. Again ~he non-adsorbe~
fraction i~ collected. This fraction i~ dialysed to
remove the PB, etha~ol precipitated to concentrate
the nucelic acid and redissolved in wat r. This
solution is treated wi~h NaOH, a3 described earlier,
in order to remove the human R-RNA. After neutralizatio^.,
addition o~ glycogen carrier, and concentration by ethanol
precipitat~on, the 3H-cDNA is redissolved in a mall
volume of wAter. Th~ 3H-cDNA preparation iQ c.omplemen-
: tary to M homini~ R-RNA but is not com~lemeneary to
human R RNA.
Hybridization of Selected 4H-cDNA with
R-RNA from Di~ferent So~rce
The production of the select d 'H-cDNA probe allows
the d~tectio~ of bacteria, including member of t~e Class
~: 15 Mollicuee~ in mammal~an tisque culture cell~ and ma~mal-
ian tissues b~ detecting the pre3ence of bactPrial ~-RNA
: by nucleic acid hybridizatlon. A nece-~ary requir2ment
oX such a tes~ hat the seleceed probe must no~
hybridize to R-RNA fro~ mammalian cell~ whlch do no~
contain bacteria. That thi~ requirement i8 ~et is shown
in Table 3V.
Tabl~ 3, parts II and III shown that the probe will
detect al~ ~embers of the class Mollicuee~ and should
detec~ all type~ of bac~ria. For example, Le~ionella_p
25 and E. coli and B8CillUJ sub~ilis are repr~sentatives
: of very differen~ bacterial type~ ~nd the probe hybridizes
~with R RNA ~rom each. of ehe~e ~ype Evolu~lonasy con-
s~derations indica~e that.thi~ probe will hybridize to
. ~ ~
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39~7
-65-
R-RNA from vlrtually any known or unknown bac~eria.
Th~q i~- due ~o the extrerlle coneer~ation of the R-RNA
nucleotide .~equence during evolution.
This ~elected probe is u3eful for detecting the
presenoe of a specific Clas3 of bacteria, Mollicute~,
in tissue culture rell~. In most tissue culture cells
antibiotlc~ are present in the growth ~ed~um and this
prevents the growth of virtually all bacteria bu~
member~ of ehe Class Mollicutes. Thu~ any cont~mination
of a tissue culture preparatlon i~ almo~t certain to be
due to a member of the (::lae~ Mollicutes.
An ~poratnad aspect i3 the ability to determine
the number of organ:l.sms pre~ent. In mo~t oases, cell
l~ne3 and ~heir product~ are di~carded when cell~ are
shown, by prior are ~ethod3, to b~ contaminated. The
ability to quantita~e the~e organism~ makes it possible
~co ~ake judgements as to the ~everity of any effectc
tue eo contaminaelc~n. The degree of a contaminatlon may
be very ligh'c, ant only one organis~ per 1000 cell3 present.
~i~ level of conta~lnation would have very litele effeo~
~: 20 on the cell~ and i~ IQany cituations the cell prQducts
neet not be tiscarded. l~he deci~io~ rQi8ht be ~de to
s,~ta~n val~able cell lines until they beco~e more
heavily cont~inaeet. Quantatiti~re considerations are
:- impo~tane for 3utging the importance of any kind of a
:~: 30 bacterial conta2alnation.
.
'~ '
7~39
-66-
TABLE 3
Hybridizat~on of Selected Mollicutes 3H-cDNA
with R-~A from Widelv Differen~ Sources
Percent ~ybridizatic~
So~ce of R-RNA
I. Con~:rolA. N~ R RNA added, ~ 1
Ex~er~tRSelf Reacticm of
3H~
B. ~ck R-RNA i~ol2tia~
C. E~an c~ll R~ 1~ to be 97%
coneaminatet with
M. h~inis R~
ybridizatialA. M~-Q of the Order of
of 3~ cDNA with ~f~rc~ ales
of t:axo~ ( ecr-~ ) 97%
` ~ cute9 - -~ 2~ ~plasma salivarius
-- ~ ect~ 93Z
3. ~e~ ~W'W'W
( ~CtS pl88) 84Z
:: :
4- ' w~W~ i' W'W
ir~ect~ ~ce) 82
B. ~ers of the Order
wh_ulcew
o e
ect~ c ows, bi~ , dogs,
~ : ; hcn~;e cat R, m~e, 3heep,
pig8j ~)t pr~a~es) 52~
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Percenc
~ybridizati~
of H-cD~A
with
S~ce of R-RNA R-RNA
II .Cl~nt ' d 2 . AcholePlasma
laidIawii
( isolate ~2) 53 %
C. 2~ers of ~he
Order
.~ SDirOP1aS
. ~ ta eae
i 1. $~ (infect~
insects and
.~ 15 nic~) 69 Z
2. E~ney bee
(isolated fram
honey bee) 68 Z
~: 3. Cac~s (isolaced
: 20 frw~ cactu~) ~ 71
4. Corn Stunt
(isola~ed from
~: CorTI) 69 %
. ~ :
5. Corn Sh~nt
(isolaeed ~ran
~sect) 65 ~ %
. ~
ybri~tion of A. ~5err~er of the ~amily
3H cDN~ w:Lth R RNA Enterobacteraceae
fr~ o~hçr :type5 0~ 1. Escherischia coli
:30 : :b æ teria (t~ ~infect5 m~mmals) 52
; ClaJ~ ~ ) B. ~emb6r of the F~mi}y
1. Legionella:
: : pneumoph~la
(infec~ man) > 28
, ~
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~78C387
-68-
Percent
~ybridizatior
of H-cD~
with
~ce of R-RNA R-RNA
III. C~n~'d C.~rr~er of the
Fami ly
crococcaceae
1. Micrococcus
luteus 50-60 %
2. Sea~lococcu~
auretls 50 Z
D. M~er of t~`te Family
La~tobacillaccae
1. streptococ~Ls
~:~ ~ 50 %
E. Y~er of ~he Family
; ~ Bacillaceae
: 1. Bacillus
~: 2~ su~tilis 40
n. ~ybrid~ Atian of
cD~ wi~h R~ 2
Yeast ~ %
- ~ ~
,
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,
.
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:1~7~39~7
-69-
TABLE 3 ~Con~'d~
;
Percent
~ybridization
of H-cDNA
So~Drce of R-RMA ~ith R-~A
V. Hybridization ~ (pr;~ee) 1 Z
R-RNA from
~ls and ~*use (rodent) 1 %
a bird Rat (rod~t) 1 %
~ ~amster (rod~t) 1 %
;~ Rabbit (lagw7Drph3 1 %
ick~n ~avian) 1 %
Excess R-RNA hybridization~ are don:e at 68 C, 0 . 48 M
15 PB. Hybridization a~say~ are don~ with hydroxyapatite
ae 67 C in 0.14 ~ P13, 0.005Z sodium dodecyl sulfatP.
The hybridiza~ion exposure is sufficient to ensure
c:omplete reaction of he ~H-cDNA with nuclear R-RNA
~: or or mitochondrial R-RNA. Nor~ bacterial R~RNA Cot ' S
;~ ~;: 20 ~ of ~t : least 2 x 103 are reached in the ca~e of the
~: mammals and bird. A non-speciL'ic sig~al of 1~2 percent
has~ been substracted from the hybridization values
prese~tet abov~
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Quanti~cation of R-RNA by Nucle~ c
Acid HybrLdization __ _
The amount of bacterial R-RNA present in a sample
can be deeermined by measuring the kinetics of hybridi-
5 zation of the selected 3H-c-DNA probe with the RNA
i301ated fro~ a tissue and comparing these kinetics to
tho.~e of a known ~andard mixture. This can be done
even in the pre~ence of a large exce~s of mamma~ian
cell R-RNA since the probe does not hybridize with
thi~ R-RNA (see Table 3, V).
For measuring the kinetics, the hybridization
mixture~ contain, 10 5 to 10 4 micrograms of 3H-cDNA
and 1 to 103 microgram of purified sample RNA in
0.01 to 0.1 ~l of ~.48 M PB . This mixture i~ incubated
at 68 C and aliquot~ are removed, dilutet to 0.14 M
PB and assayed for hybridization at various times after
the initiation of the reaction. Hybridization assays
are performed using hydroxyapatite as de~cribet earlier.
The re3ult~ obtained are compared to the hybridization
of the probe reacted with ~tandard RNAs con~aining known
amounts of bacterial R-RNA. These standards are mixtures
of mammall~n c~ll RNA and known amount~ of a ~pecific
bacterial R-RN~.
:
Detecsion and Quantitation of MemberY of the
Class Mollicute~ in Tissue Culture Cell~
Table ~ pre~ent3 daea obtained by hybridizing the
selected probe with RNA i~olated (a-~ described earlier~
from three differe~t ti~sue culture cell sampels. Only
cell line number 3 i detectably contaminated and the
kinetic~ of the reaction indicated that aboue 5 x 10
bacterial cell~ are presen~ in ~he ti~ue culture cell~
. ~
1.~78~387
71-
TABL~ 4
Detection and_~antitation of Mollicute3 in Tissue Culture C lls
Hybridl- Hybridization
Time of ~-cDNA Num~er of Bacteria
5Cell ll~e (hours) with RNA DYtected
.. . ~ .... _ . _
1. 44-2C (rat) 17 1 None detected
1 Ncne detected
2. P388 D~M
(mouse) 1.1 1 Nbne deteceed
22.5 . 1 None detected
3. P388 DlC 7
(mouse) 0.025 20 5 x 10
16.2 78 (ab~ue 1 M~llicute
per mammaLian Ln
cell)
.
,''
Excess R-RNA Hybridizations are done at 6B C in 0 . 48
15: M P33 in a vo}ume of 0.01 to 0.04 ml. Each mixture
contains 2 x 105 micrograms of 3~-cDNA probe and
: 50 200 micrograsl-s of sampl~ RNA.
~: :
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~ 78~8
-72-
~he foll~wing example i9 another embodiment of
the method of ~y inYention, used for detecting very
small numbers, even one trypanosome, ln ~he pre~ence
of a large number of blood cell~.
The detection of trypanoso~es is important since
certain members of the protozoan group TrYPanosoma are
pathogenic for humans, causing d~seases that include
East African c1eeping sickness, We~t African sleeping
sickne~s, and South Amerlcan trypanosomiasis. These
organisms are large and have varying characteristic
shape , depending on the stage of the life cycle. Prior
art methods rely mainly on serologic, differential
taining coupled w~th microscopic examination and animal
inoculation prooedureY for detecting these organisms in
hu~ans. The serod~agnostic method~ ~ary in ensiti~ity
and specificity and may be difficult to interpret. The
microseopic method~ are most used, however small number3
of the trypanosome3 are cten difficult to detect in
the presence of larg~ nt.~bers of blood cells. Animal
inoculation 1~ a long and co tly procedure.
. The embodl~ent of ehe in~en~ion set forth in the
following ~xa~ple 1~ a method which make-R it relarively
ea~y to detect the presence of one trypanoso~e even
when co-pre~en~ w~h a large number of ~lood cell~.
,~
~
Radioactive DNA complementary (3H-cDNAo to try~an-
osoma bruee~ R-RNA iq produced in the ~ame w y a~
.:~
~, ~
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:,
,f,,~
,j.
~ ~:7~ 7
- 7 3 -
M. homini.~ 3H-cDNA, which is de3cribed above in detail,
. .
execpt that ~r~2~no~ma b R-RNA iq used a~ a template.
Selectlon of Trypano30me 3H-cDNA Which is Co~plementary
to Trypanoqome R-RNA but is not Com~lementary to ~UE~ R-
Thi~ i~ done in the same way as described earlier
for M. hominis except that TrypanoQoma b. H-cDNA is
hybridized to the human R-RNA.
USQ of Selected Trypanosome 3H-cDNA ~o Detect
and Ouantitate n~anos ~ s ~ ~En Tis ~ or ~uid
The production of the selected 3H-cDNA probe allows
the detection and quantitation of trypanoso~es in human
samples by detecting the presence of trypano~ome R-RNA.
A necassary requirement of such a test is that the
selected probe mu t noe hybridize to R RNA from human
cells which do no~ contain trypanosomes. Tabl~ 5 shown
that this requirem~nt i~ met.
.~
_ 7 4 ~ 7~39~7
TABLE 5
Eiybridization of Selected Trypanosoma brucei
,,
'H-cDNA with R-RMA from Different Source3
_ _ _ _
Percent Hybridization
R-RNA Source ofH-cDNA with R-RNA_
.~ 5 No R~IA added 1 %
Trypanosom_ brucei R-RNA 98 %
Bacterial ~Mycoplasma hominis )
R-RNA 1
Human R-RNA 1
Human R-RNA known to be
coneaminated with Trypanosome 98 %
,
Exce.~s R-RNA hybridizations are done at 65 C in 0.48
M PB~ Reactions are run fo~ 24 hour~ ant the hybridi-
`~ : 15 zat~on exposure is ~ufficient to ensure complete reaction
of the human nuclear or ~itochondrial R-RNAs and the
~: ~ bacterial R-RNA. Hybridiza~ion assays are done wieh
:~ hydroxyapa~ite at 72 C in 0 .14 M PB, 0 . 005~ sodium
dodecyl sulfate.
~ .
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.
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One illus~rative probe which I have prepared i~
specific only for member~ of the Genu~ Le~ionella.
The probe hybridizes to greater than fi~ty percent
with nuclelc acid~ from diverse members of the
Genus Le~ionella, and does not hybridize signif-
ioantly wlth nucleic acids from mammals, yea3t and
a variety of widely diverse baeterial strains (Table
8). The probe hybridize~ well even with Legianella
species such as L. pneumo~hila and L. icdadei
which show little or no bulk DNA relatedne~s. Other
known ~ species can be detected by thi~
probe u~ed in Table 6; as listed ~n Table ~ All
of the known Legionella specie~ (ehus far 23 differen~
~pecies) ha~e been examin~d and all can be speclfi-
cally d~tected withthe probe used in Table 6.
: The ~pecif~city of th~s probe make i~ pos~ible
to detect and quantitate the presence of ~egionella
~pecies, eYen in ~he pre~enee of large number~ of
non-related bacterial or mammaliam cell~. Thus, liver
cell from a L. ~ oph~la infected hamster was
assayed for the pre~ence and number of Legi nella
organis~s by u~ing the specific probe and well established
~: n~cleic acid hybrld~zation procedure-~. The liver had
preYiou~ly bee~ a~sayed by a ~icrobiological growth te~t
.: 25 whlch inticatet tha~ abou~ 107 ~ organis~s per
: gram were prasent in the infeceed livex. Nucleic acid
hybridization analy~i3 indicated about 1 - 2 x 108
Legionella organi-qms per gra~ of liver. The~e results
~ug~e3t9 t~at the~a~ing ef~iciency ~n the growth test
i8 about 5 ^ 10 percent.
:
.,~:
`:
-76~ 7~
The specific probe allows the highly sensitive
and rapid detection of Le~ionella org ~ ~ even in
the presence of large numberc of mammalian cells.
In an assay which took les~ than 1 day the probe easily
detected the presence of about 400 Le~ionella organ sms
which were mixed with 0.4 mg of liver (about 2 x 10
cells).
`: :
TABLE 6
Hybridizat~on of Legionella Speclfic Probe With
Nucl~ic Acits from Widely Different Sources
Normalized
Nucle~c Acid Percent Probe
' 50urce Hybridized
I. Con~ol~ 1~ No n~cleic ~id
;~ 15 2) Mbck nuc~ ~ acid Isolat
3) L ~neum. ~ ected
issue 100 (Ac~
percent ~ 81)
II. Le~a~l~-
:~ . ~ ~ 1) z~Enii ~GA) ~ 59
2) L. ~ff~ (TEX-K~j ?50
___
~: :
3) L. ~æ~E~i (LS-13) ?50
4) L. jor~ (~ 40) ~50
S~ 50
:~ 6j L. m:lcdadai (H~) ~ 50
7) L. M~ ~ 50
8) L. oao~ J (~i ~ 10) > 50
, ~ ~
'
~77
~7~3~38
~E 6 (Cont'd)
No~malized
~clel~ id Percent Probe
Source
~ 5 ~) L. pneu DphilA (P~A 1) 100
; 10) L. ~ans~ 2 > 50
11) L. SC-32C-CB > 50
III . Other 1) Aec r~as c~r~Dhila
Bac~erial
Specie 2~ B s~b~ilis 1
3) !~
; 43 ~5~ ~!a~ 1
.: 5) 1:. ~oli 1
6~ Fl~bacteriull bre~
, ~ ~
7) ~ " ~ 1
8) " ~iDgosept~
9~ " ~ltivar~
:18) Ps~ ~ al~es 1
",`, '~ ~i; ~
;:' ~ ~ : :
, ' '~ ' ' '
'i` ' `:
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7~187
Nor~ 1 ized
Source of Percen~ Probe
Nucleic Acid HYbr dized
III. Other 19) Vlbrio El Tor
Bacterial
Specieq 20) Mycoplasma homini~
( cont ' d)
21) " ~
22) " salivarium
23~ Acholeplas~a Laidlawii,
2 4 ) Sp iro~ 1 a~ma SMCA
25) " corn qtunt
26) " honey bee
27) " cactu~ 1
15 .IV. Yeast S. cer~r 1
.
V. Mam~als Human
Hams ter
Mou3e
~:: Excess R-~NA hybridi2ation~ are done a~ 76 ~, 0.48 M PB.
20 Hybridiza ion a~say~ are done with ~ydroxyapatite at 72 C
in 0.14 MPB, O.OOS% sodlum dod2cyl ~ulfate. The hybriti-
zat~or. e~Cpocure i~ qufficient ~co ensure compl~ee reaction
q
of th~ JH-cDNA with nuclear R-RNA or for mi~ochondrial
:~ ~ . R~RNA. Non-bacterlal R-RNA Cot ' ~ of at lea~t 2 x 10'
~ 25 ar~ reached in the ca~e of ~he mammals and bird-~.
. ~
~, ~
:: :
1~78{3B7
79
TA:13LE 7
Oth~r Legioneila ~pecie~ Which Can Be Detected
B~ Specific Nucleic Acid Probe of Table 7
SDecies
_ WA-316
L. WO-44-31:: (L. feeleii)
L_ Phoenix- l_ _
WA- 2 7 0
L. PF-209C-C
L. SC 65C3 (ORW)
L. Jamestown 26Gl-E2
L . MSH- 4
L. ans~
: ` L. SC-I8-C9
:: ,
L. SC-63~C7
: I, 81-7l6
:
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1~78~7
-80-
Example 3: Production of a Probe Which Will Hybridize
0~ly to R-RNA cm ~rs of ~ ~u~ Le~ionel~
Pro~t~ of Ra~ac~ive DNA ~x~l~mentary
to Le~ella R-RNA _ _ _
R RNA from the ~pecies Legionella pneumo~hila is
u~ed as a templ~te to synthesize marked (radioactive)
cDNA (eomplementary DNA) complementary to Legionella
: pneumo~hila R-RNA. This cDNA is produces by utili~ing
the abili~y of an enzyme, reverse transc ptase, t~
utilize R-RNA a a template and produce H-cDNA.comyle-
mentary to R-RNA. T~is is done in the s~me way a~
described for producing M. hominls 3H-cDNA excep~ that
R-RNA fro~ Legionella ~_eumophila i~ u ed as ~ template.
lS Selection of Radioactive Probe which Hybritize
~: only to R- ~ ember~ of the Genu~_LeRionella
The purified H-cDNA ~-~ fract~onatet by hybridizing
it with a 8reat excess of R-RNA rom E. coli, Acheola~lasma
2Q laidLswaii and Prsviden~ia stuar~ii. The hybrid~zation
~xit~re consi3t~ of 0.o5 - 1 micrograms of 3H^cDNA and 20
microgr2~s of each bac~erial R-RNA in 1 ml of 0.48 M PB.
~ Thi~ mixture 1~ ~ncubatet a~ 76 C for 1 hour and ~he
: ~ixture i~ then diluted to 0.14 M P8 and pa ~ed over HA
equilDrated eo 72 C, 0.14 M PB. The fractio~ of 3H-cDNA
: wh~ch doe~ ~ot adsorb to the HA (~.e., the H-cDNA no~
hybridiz~d to the R-RNA) i~ collected. Thi8 raction is
` then passed o~er HA under the ~ame condi~ions as aboYe
:~: . and again the non-ad~orbed fraction is collected. Thi
3H cDNA is ~hen concenerated and aga$n hybridized with
~ ~ bae~erial R-RNA ~ describet above. The non-adsorbed
.~ fract~on i3 collected and co~centrated and ~h~n hybridized
.~ ..
:
~'
.~ .
.,
~ 7~ ~8
81~
with bacterial R-RNA for a third time as described
above and fractionated on HA a~ above. The non-adsorbed
fraction is collected, base treated to remove any R-RNA
present and concentrated into water. This 3H-cDNA pre-
paratlon will hybridize to any me~ber of the Legionella
genu~ and will not hybridize to R-RNAs from other sources.
Hybridization of Leg~onella specific 3H~cDNA Probe
Wi R-RNA and R-RNA Genes from Different Source
The selected probe allow3 the d~tection o~ any
me~ber of the genu-R ~ in a ~a~ple by detocting
the presence of ~ R-RNA by nucleic acid
hybridizatlon. A neces3ary require~ent of 3U h a ~es~
i~ that ~he ~ pec~fic probe ~U3t not hybridize
to R-RNA from o~her ource~.
Q~aneitation of Le~ionella R-RNA by Nucle~c_ id Hybrid~7ation
The amount of bacteria~ R-RNA present in a sample
can be tetermi~ed b~ ~easuring the k~net~c~ of hybridi-
za~ion of the sel~cted 3~-cDNA probe wlth the RNA
isolated fro~ a ti-~ue ~ample and comFaring these kinetics
to tho e o~ a k~own ~tandard ~ixture. Thi~ can be done
even in ehe pre ence of a large exces~ of m G alian ce~l
R- ~ ince the p~obe doe~ not hybrit~z~ with this R-RNA.
For mea~uring ~he kinetic~, the hybsidization
: mix ure may conta~, for exam~le, 10 5 to 10 4 micrograms
:~ of H-cDNA ant 0.01 to 10 microgram~ of purifi~d sæmple
RNA in 0.01 to 0.1 ml of 0.48 M PB. Thi~ ~ixture i~
in~bated at 76 C and aliquot~ are removed, dilutet to
~ .
: '
~ ~ .
~ ~ ~8 ~7
-82-
0.14 M PB and assayed for hybridization at various
time~ after the initiation of the reaction. Hybridi-
zation assay are perfromed using hydroxyapatite
as described earlier. The results
obtained are compared to the hybridization kinetics
~: of the probe reacted with standard RNAs containing
known amounts of bacterial R-RNAo The~e standards
are mixtures of mammalian cell RNA and known amc~unts
of a specific bacterial R-RNA.
Table 8 present~ data on the quantitation of
Legionella pn umophila present in water samples and
in an infeceed ham~ter liver sample. Th~ water samples
and the liver sample-Y were titered for the presence of
L. pneum by standard quant~tative growth assays at the
Cen~er for Di~eaqe Control in Atlanta, Georgia.
TABLE 8
: M~d by ~sured by E~CPSS R-RN~
a Grow~h Assay Nucleic A~id HYbridization
L. hila bacteria 107 bacteria 1 - 2 x 108 bacter~a
per ~ ected gra~ liYer ~ lr~er
. ~ hamSt:OE live~
:: n o
L. ~ bæterl~ 1.5 x 10 bacter~a 2;1 x 10U bacteria
~ ~er o water ~le --~ ml
i'~ .
Excc~ Prob~ M-Cbod
The amoun~ o~'bacterial R-R~A pre.~ent ~n a sample
can al50 be measured by doing hybridization under con-
, :; . :'~ ..
.
~ . ' .
,~
. .
: . .
''~
:: `
, :
~ ,78 ~87
-~3-
dition~ where there $~ an exce~ of the Le~io~ella
speciic 3H-cDNA probe, relative to the amount of
Le~ionella R-RNA prese~t. Thi~ ~ixture iq hybridized
to completion. At this point each Le~io~ella R-RNA
molecule pre~ent in the sample i~ saturated with probe
molecules, By compar~ng the amount of probe hybridized
to the R-RNA to an appropriately con tructed standard
calibration curve, the amount of R-RNA in a samp~e can
be deter~nined. A goot e~ti&ate of the total num.ber
10 of ~ Pneumoph-ila bacteria present: in the sample
can ~hen be calculated by knowing the a~verage number of
R-RNA molecules per L. pn~u~nophlla bacterium.
Table 9 present~ data on ehe quantitation of L.
pneum~hila presen~ in water sample~ as determined by
15 the exces3 prob~ accelerated hybridization rate - enzyme-
detergent-sample method described in deta~l in a la~er
section. The water samples were ti~er~d for ehe presence
of L pneumophila by ~tantard quaneitati~e growth assays.
The~e assayq take day~ to complete while the hybridization
as~ay take~ about 1 hour.
TABLE 9
'~'
~ Mea~ured byMea~ured by the Exces
~'
'
L ~ 1.5 x 108 bactcri1 2.2 x 10~ bæter~a
25 bacterial per ~ 3r-~
water sample
.
;
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.~
'::
.::
~.
..
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7~38
-84 -
obe S~ecific On1Y for R-RNA from M~ers of t~ ~ Le~ionella
A. wlwi1 cf ~a~er a~: Accelerated ~ybridization Rate ~thod
1. Preparatian o 5a~1e and ~bridizati~ Inc~bation Mixture:
Mix in the following order æ quiclcly a~ pos3ible.
S a) 9 mlcroliters of sa~le
b) 2 ;rrolit~rs of enz~-detergent solution coT~tainin~:
5 milligrams/~nl Prote~se K, 0.5 M Tris ~pH ~ 8.1),
8% sodium dodec~l sulfate (SDS), 4% sodiu~ sarcosinate,
0 . 25 M Na~l, 0 ~ 016 M EI~A, 0 . 016 EOEA
c) 1 ~ierolit~ of probe d~ssalv~d in water
d) 20 micxolit~ of 4.8 1!~ PB
~: 2. ~a~e the mi~e at 76 for an ~ppropria~e ti~ so
that *le hybrldizatior reaction i~ c~lete.
3. The h~b~idiza~ assay is perfonE~d as follows:
a) A~d the ina~bation mixn~re to one ~1 o~ a r~
tes~ature solution containing: 0 . 05 gram~
h~apa ~te (HA), O.054 M PB, O.02Z Zwitte~-
gOElt 14 (~i~chem) (hereinafter referred to
a~ Z-14)
b~ Sh~ae ~ mlci~e ~or 30 sect~nds at ro~ te¢~3erature,
add 5 ~1 0.14 M PB, 0.022 Z-14, ~nd inn~ate ~ Dix~re
at 72 C ~ar 2 m~sute~.
G) Centrifilg~ the soluticJn to pellet *~ ~. All
centri~i~ æe d~ne at r~ te~era~re.
~S and ~e the liquid fracti~ wash
tl.
' ::
. ,
~` :
'~:
_ ~ 5 ~ 7
d) Add 6 ml of 0.14 M PB, 0.02Z Z-14
~olution to the pellet. Re.~uspend
th~ ~ pellet by vortexlng it.
Centrifuge to pellet the HA and decant
S and Rave the liquid fraction. Thi~ is
wa~h ~ 2.
e) Repeat step d. Thia re~ults in wash ~3.
f) Add 6 ml 0 . 03 ant resuspend the HA pe~llet
by vortexing. Centrifuge the suspes~sion
io to pelle~ the HA and decan~ the liquid
and a~say :i.t for the pre~ence of the probe.
Thl~ fract~ on ontain3 the hybrldized probe,
if any is pre sent .
~: It i~ not necessary to elute the hybrid~zet probe fron~
. 15 the HA under certa~n conditions. Thus, if the probe ismarked with a marker which can be detectèd in the presence
of HA, the pellet from 4tep e can be assayed tirec~ly for
~he probe. In the case of a marlcer suoh a-~ Iodine-125
~ ~ ~ the tube containing the HA pellet can be placed direc~ly
: :~ 20 in~o a gam~a detec~ion machine.
:. ~
Other modifica ionQ can al~o be made to make the
:~; ; te~c fa~t~r and ea~ier. Thu~, the volume and asnount
of ~ u~et caR be ~calet down, arld the nu~b~r of waqhe~
can also be ~ r~duced, tepending on ~he ~ituation. In
~: 30: ~ oeher in3tance~ ~ t ~ay be de~irable ~co increase the
Yolu~e ~of ~ :or slu~beI of wa~he~ variety of salts
other: ~han ~odiu~ pho~pha~e, and other detergentQ can
alJo be u~ed~ he assay.
.. ~
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~1 ~7~ 37
-86-
B. Arlalysi~ of a Liquid Sa~: Standar~ion Rate ~thod
1. Preparatiorl of Sa~le ~nd ~ridization Irn~batior~ mix.
Mix in the ~ollowing order and as qu$ckly as possible.
a~ 14 mlcroliters of sa~le
b3 2 microliters o~ e~-detergent solution described
in A.
c) 1 microliter of probe
d) 3 microliterq of 3.2 M PB, 0.03 M EDIA, O.û3 M EOE~
2. In~ubate the n~ure ae 76 C for an ~ropriate tise
so that ~ybridization will ccuple~e.
3. l~ br~dizati~n assa~ i3 perfor~d as follows:
a) A~d ~he in~atian m~ to 1 ~il of a solutia~ cc~ntaining
0.14 M PB, 0.02Z Z-14, Q.Q5 ~am~ of HA.
b) Fra~ point on the protocol is identical to that
: ~ 15 desc~ ed in A.
`: : C ~L~ c~l~ ~
~; A 10 percene liver hc~glate in water is used as ~e tissue sa~le.
1. Prep~atian o~ Sa:~}e and ~nQibation Mix.
~y a~ possible ~n *~e follawing order.
~ a~ 8 microl~ of sa~le ~10~ 7er ~genate)
b) 3 ~ll~lt~s o~ enzy~-detergent ~c cocltaining:
8%~ i.~odiun sa:rco4inate9 0.15 M~NaCl, .016
M E~rAg 0.016 M EOEA, 0.38 M Tri8 (pH a 8.~) ~ 195
millig~fisd of Prana~e.
25 ~ : c) l ~oli~ of probe spe~fic only f~ ~ R-R~.
d) 20 D~s:roli~rs of 4.8 M PB.
,.
:
. ~ .
.,,~ ~ .
: ~
~ 78~7
-87-
2. Incubate the mixture t 76 for an appropriaee
time to ensure that the hybridization reaction
is com~lete.
: 3. The hybridization assay is performed as described
in the section on analysls of a water`sample;
: Accelerated Rate Method.
D. ~h~: Standard ~oridi2ation ~ate ~thod
~ ..
A lO percent liver ha~ge~ate in water used as the s ~ le.
1. Preparation o ehe ~le ~nd ~ ation M~x.
Mix as qui ~ y ~s po~sible in ~ foll ~ order.
~; a) 12 m~ollter~ of s ~ le.
b) 4 ~dsroliter~ of ~ detergent solution descr ~ d
: in B~
; : c) 1 md~roli ~ of probe ~ec~fic only for
: R-~M~.
.d) 3 ~ liter~ of 3.2 M PB, 0.03 M EDrA, Q.03 M EGEA.
: :15 2. ~ ate ~ ~ixe or an ~ ropriate t ~ at 76~ C.
3. The hyJrs~zat~on a~say is perf~æd a~ foll ~ .
a) adt ~ ~ ation m~x to 1 ~1 of a ~olutian
c~nCi~ng 0.14 M PB, 0.02~ Z-14, 0.05 gr ~ HA.
: b) ~ron~ 3 point ~ as~ay ~ ~t~cal to ~ t
~cr ~ d in A.
A ~ e de~led ~escription of n~cleis acid ~ ion te~t Oo
:: detect ~Ek~ E~ bact~ n water and live~ 5 ~ les
pre~ d below.
~ ~ :
.
,
.: :
" ~ :
,:
~1 ~78987
-88-
Examp le 4
Rap~ ~iti~re Detect~on of Le~e~la B~teria in Water
Sanrle: Aceelerated }~te ~ethod
1~ Ihe ~ollow~ng componenss were mixed as quiekly
as pos s ibl e and in the fo llowing crder ~
a) 4 . 5 microliterq of a water sample con~aining
about 105 ~ ~ baec.erial
per ml . The number o bac ceria in t~e water
wa determ~ned at the Center of Disease Control
in Atlanta by a grow h tes~.
b) 1 microliter of the enzyme-detergerlt solutio~
de~ribed in A.
c~ O . 5 ~icroliters of ~ spec~ :fic probe .
The amount of probe equalled ~ 5 x 10 6
micrograsn3.
d) 10 microliters of 4 . 8 M P~ .
~: Asse~bling the hybridization mixture about 2 minutes.
'
2. Incubat~ the hyrbidization m~x'cure for 36 minutes
at 76 C .
:
3. Perfo2~ the hy~idization a3say a~ de~cribed in A.
This t~ok about 5 ~inu~ce-4.
4. As~ay ~ch2 fractiolls for ~he p~e~ence of the probe.
Tlli9 took ~ou~ 10 minute~. ~
.
The n~ber o~ l;egionella bac~er~a presen in the
hybridizae~on mixture wa~ abou~c 500. Thi~ number of
,.
organisms was ~det~ct~d and quan~itated in a~out one
..
.
; .~
,.- ~ ~ ....
.
~1 ~78~38'7
-89-
hours, from ~eart to finiqh, with the use of less than
10 5 microgram~ of probe. Twenty-three percent of the -
probe hy~rid~d with ~e Le~ione~a R-RNA in thi~ test
which wa.q de3igned to be an exces~ probe hybridization
test. Control te~e~ were done under the ~ame conditions,
one with no bacteria added, and one with about 105 E.
coli bacteria present in the hybridization mix. In
both cases only 1-2 percent of the probe behaved~as
if it were hybridized.
The above te~t can be modified to acsay larger volumes
for the presence of Le~ionella bacteria. Thus, one ~1
of a water sample containing 104 e~ionella bacteria
per ml wa~ centrifuged for 3Q minute~ ~o pellet the
bacteria. A small amount of enzy~e-detergent was added
to thepellet and a hybridization test was performed on
this mixture u ing the accelerated rate method and the
LeRionella bacteria were readily detected. Much larger
volumes of .qa~ple can be c~ntrifugcd and other methods
of concentrat~ng the bacteria, includ~ng membrane filtra-
tion, can al~o be u~et. These modification make itpossible ~o detect a s~all number of bacteria in a lar~e
: sa~le volume. Air Qample can also b~ concen~rated by
~: methods, including ~embrane filtration methods, ant
small number9 of bacteria can be detected in large
volumes of air sample.
Example 5
=le
}. The follawlng cc~ts ~ere mixed a~ quiclcly a~ possible
;~ ~n the fo~l~ ord~.
':~
"~:
.
~ -
~.~ 7~987
-90-
a) 4 microliters of a 10 percent liver homogenate
of a hamster liver infected with Le~ionella
pneumcphila. This i9 equivalent to 400
micrograms of liver or about 6 x 104 liver
cells. About 750 Le~ionelLa E~
were present in this sample.
b) 4 microlieers of an enzyme-detergent solueion
: composed of: 45 milligrams/ml Proteinase K,
8~ SDS, 4% sodium sarcosinate, 0.5 M Tris
(pH = 8.2), 0.008 M EDTA, 0.~08 M EGTA,
0.25 ~ Nacl.
c) 4 microliters of Le~ionella specific probe.
The quan~ity of probe was about 10 micro- -
grams.
~:
: ~ 15 2. Incubate ~he hybridization mixture at 76 C for 3
hours.
3. Perform the hybritization assay as described in A.
,. '
:~ 4. Assay ehe re~ult~ng fractions or the presenca of
~ probe hy~ridized ~o ~E~ R-RNA.
.. , ~
: 2~0 The nu~b~ o~ ~ bacteria present in the
hybrldization m~xture was about 7~0 and the amount of
: : R-RNA presene ~n this:n~ber o~ Le~__nelLa cells is
about 1.1 x 10 5 microgram~. The number of liver ceLls
: present wa~about 6 x 104 and ehe amount of liver R-RNA
:pre5ent:wa3 about one microgram. Ten percent of the
sp~cific probe hybridi~ed. Control tests were
,, ~
::
"
;~,
b, ~
)'';
- 9 1 -
done wi~h unifected liver in the same manner and less
than one percent of t~e probe behaved as if hybridized.
Examples 4 and 5 illustrat_ only two of the ~any possible
conf~guration~ for such a te~t. Test3 utilizing different
volumes, salt~, detergents, probes, sample types, prote-
olyti~ enzyme~, amounts of ~A, incubation per~ods,
organism types, amounts of probe, temperatures of
incubation, ant hybridization rate accelerating sy~tems
can be succes fully utilized within the general ~o~tex~
of the test3 described here. Any of the R-RNA probes
can be used in a sy3te~ comparable to tho~e dcscribed
above. Non R-RNA probes can also be u~ed to good
effect in the~e sy3tem~ with so~e obvious ~odification~.
: For example, a teqt -~pecific for a particular DNA ~equence
; 15 in a specific organism or group of organi m~ can be done
exactly as described above if a step i.~ included which
converts the double strand DNA ~o the singel strand for~.
In other case~ different modification~ of the method
mu.~t be used. Bacteria such as Mycobacteria and Bacilli
are difficult to break open. A step which breaks open
- these bacteria mu~t then bc used in con3unction with
the method deccribed abov_. A single incubation, in
: the absence of detergents, with ~h~ enzy~e ly-~ozyme,
~:: will ~ake mo t ~aclllus bacteria su~cept~ble to lysis
-~ 25 by detergent~, or example. On the other hand, Myco-
~ baceeris ar~ very difficult to lys~ and may haYe to be
; . phy~Icall broken open before they can be tosted for.
;~ A modification of th~ above method ~an also be
~ : uset in con~unctl~n^with any ~ran3fer ~NA probe or a
:~ 30 probe specific for any other RNA present in an organism.
: .
';
.
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.
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,~
,. ~
,~ ~
,,
,.~
~78~7
~92-
A qtep designed to concentrace small numbers
of bacteria or other cells out of large volumes of
sample~ ~uch as alr or liquid can al~o be used in
con~unction with the hybridization te~t to detect
mo~t other bacterial organi~ or other types of
organisms .
While I ha~e described above, in detail, the
produc~ion and ~se of a nucleie acid probe which~
hybridizes only to nucleic acids fro~ me~ber~ of.
the Genu3 Legionella, it wlll be readily apparent
to thoqe skilled ~n the art fro~ that exa~ple and
the others, that o~her probes can be produced, based
on ~he procedures illustrated above. Thu~ th~ method
uset to produce quch other probes would be a~ follow~:
1. Produce market nucleic ac~d complementary to
the R~RNA of a member of the gro~ of interest.
2. Hybridize thi~ DNA to the R-RNA from a member
of ~h~ group of orga~is~s evolutionarily most
closely related to the group of organisms for
which the probe i pecific. Select the fraction
: of the mar~et nucleci acid which, at a specific
criterion doe3 no~ hybridize to R-RNA fro~ a
: me~ber of ~hi~ closest related group of organ~
i~3. Th~3 fractio~ is speclfic for the organ-
is~ group o~ interest.
Example~ ~f the~e are:
a. The produc~ion of a market probe which hybridizes
: only w~t~ R-RNA fro~ a member of the bacterial
: : Genu~ and toes not hybridize with
~ 30 R-RNA other ource~.
,~ ~
`~:
.
, : ~;,
'
~ 7 8 ~87
-93-
b. The production of a marked probe which hybridizes
only with R-~NA from a member of the bacteri~l
Genu~ M~coplasma and does not hybridize with
R-RNA from other sources.
c. The production of a marked probe which hybridizes
only with R-RNA from a member of the bacterial
Family Enterbacteriaceae and doe~ not hybridize
wi~h R-RNA from other source~.
d. The produceion of a marked probe which.hybridizes
only with R-RNA from a member of the anaerobic
group of bacteria and does not hybridize wi~h
R-RNA from other sourres.
. The production of a marked probe which hybridizes
only with R-~NA from a me~ber of ~he group Fungi
and doe~ not hybritize with R-RNA from other
~ources.
f. The productlon of a marked probe which hybridizes
only wi~h R-RNA from any member of th~ Chlamydia
: group and does not hybridize with R-RNA from
other 30urce~.
g. The productlon of a marked probe which hybridizes
only with R-RNA from any member of the bact~rial
; famlly MYcobacteriaceae and doe~ not hybridize
: with:R-RNA fro~ other sources.
25 h. The production of a marked probe which hybridizes
R-RNA fro~ any living organ~sm.
The production of marked probe which hybr~ dizes
~: only w~th R-RNA from any mammal and toes no~
hybridiz~ w~th R-RN~ from other sources.
."~
, ~
,~": ~
.
:~
7~39~3'7
-94 -
Illustrative l~mbo~t of the Use of Pro~es for t-RNA to Detect,
~rntitate and Identi~ 2nis~
t-RNA probes may be used in the same mann~r as the
R-~NA. probes to detect, identify and quantitate organisms
S ant in some case~ viruses. For example, a t-RNA probe
specific for Le~ionella can be produced and used in a
manner similar to that descrlbed for the R-RNA probe
specific only for e~ionella. The ilLustrative embodi-
ment described for the Le~ionella specific R-RNA probe
thus also er~res a~ an illu tration of a t-RNA specif ic
probe.
The genes present in ;~any DNA and RNA v~ru~es include
t-RNA gene~ which are ~pecif~c for the virus.
Illustrative ~t of t}~e Use of Probe~ Speci:Eic far ~A, knRNA,
lS snR~ or~sR~ to: Deeect! Qu~titate ~d Id~ntif~ isms, Cells
~nd VL~
ProbeQ specific fos mRNA, hnRNA, snRNA or psRNA may
be usat in a manner alalogou~ to those for R-RNA arld
t-RNA to detec~ dentify and quantita~e a specific
` ~ 20 large or small group of organisms, cell~ or vlruses
in ce3.ls. S~nc~ the evolutionary con~er~Jation of the
individual gen~ which produce tho~e various RNAs varies
grea~ly it is pos~ible to produce probes wh~ch will detect
~; m~mb2r~ of very large classe~ of organ~ s~ and probes which
25 d~eect member3 of relati~ely s~all classes of organism~,
cells or viru~eq in cel}Q.
One example of highly conser~ed gene . equences are
the hiseone gene~, a fa~nily of genes pre~ent in eukaryotic
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cells. Histones are nuclear structural proteins which
are present ln all eukaryotes. The histone DNA sequences
show greae similarity even in widely diverged organisms.
Thus, the histone genes of sea urchin and man are similar
enough. to hybridize together. A probe which is specific
for a particular histone mRNA or for a population of
histone mRNA~ can detect, identify, the presence or
absence of any member of a large group of widely diverse
organisms. The sensitivity of detection of cells or
10 organisms is enhanced by the abundance of the histone
mRNAs. In ord~r to grow, a cell or organism must
synthesize histone mRNA in large amounts.
Another example invovles certain gene sequences which
code for psRNA and are not conserved during evolution.
Such gene sequences fro~ one type of organism do not
hybridize to DNA from distantly related species. A
; probe specific for one partlcular psRNA sequence or a
population of different ps~NA sequences from one organ-
is~ type or viru~ type, can be used t~o detect, quanti~ate,
and identify member~ of a small group of closely related
organis~s or a small group of closely related viruses
~: which are i~ cells.
: Another example i~ the developme~t of a prob~ specific
- for a sequence or sequence-~ o mRNA, hnRNA, snRNA or psRNA
which can be u~ed to examine body fluids for evidence of
specific cell damage and des~ructi~n. In certain dlseases
cell~ are destr~yed and their content~ including cell
nucleic acid~ ar2 ~pilled in~o the circulating blood.
Liver damage due~to hepatitis is one such situation and
:- ~ 30 i~ is known that both DNA and ~NA from liver eP.lls enters
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789~37
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the circulating blood as a result of c211 damage. A
probe can be produced which is specific fo~ an RNA
sequence or sequences which are characteristic only
of liver cells and are not present in other nor~al cell
types. The exis~ence of such RNAs is well known. This
~robe can then be used to detect, idencify, and quan~itate
liver speci~ic mR~A, hnRNA, snRNA or psRNA sequences in
blood samples by nucleic acid hybridization m~thodology
as described herein. The presence of liver damage and
10 its extent can be determined since the amount of RNA
present in the blood will reflect the extent of cell
damage.
P~obes specific for a particular ~RNA, hnRNA, snRNA
or ~sRNA sequence or sequences present only in a specific
15 - cype of liver cell can also be produced and used to de~ect
and quaneitate the presence in the blood of the RNA
sequences resulting from the damage of a specific liver
;~ cell type. Obviously the morc abundant the specific RNA
sequence in a liver cell the higher the sensitivity of
detection of ehe RNA.
Damage to any body tissue or organ (including heart,
kidney, brain, muscle, pancreas, spleen, etc.) may be
detected and quantitated in this manner. Other body
fluids including spi~al fluid and urine can also be
assayed for the presence of these specific RNAs.
A useful initial screening test for tissue damage
from any source can be done by utilizing a probe speciric
for R-RNA and examining blood or other body fluids for
the presence of R-RNA or t-RNA sequences. Quantitation
of R-RNA or t-RNA present will provide an indication as
to the extent of tissue damage without identifying the
~- source.
~:
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Another sample of the use of the nucleic acid
hybridization tests and approaches described herein
is the detection and quan~itation o E. coLi cells
containing the plasmid genes which code for the E.
c_ entertoxin pro~ei~. Such a test involves the
use of a marked nucleic acid probe complemen~ary to
the enterotoxin protein mRNA in order ~o detect and
quantita~e the presence of E. coli bac~eria containing
the enterotoxin protein mRNA. This can be accomplished
by utilizing the in solution hybridization methods
described herein.
As discussed herein bcfore the use o a probe
complementary to E. coli enterotoxin mRNA as a means
to detect and quantitate the presence of E. coli
bacterial which are producing E coli eneerotoxin
and therefore contai~ enterotoxin mRNA, by using nucleic
acid hybridization methods, has signifioant advantages
over methods such a3 de~cribed in the Falkow et al.
patene discussed earlier.
; 20 The sa~e approach as described above can be utilized
; to detect the specific gene product of a particular
microorganisal which confers resistance to a particular
antibiotic or other anti~icrobial agent. Th2 genes
which confer re~i tance to mv~t antibiotic-~ are almost
al~7ays pre~ene on pla~mids in the cell. In order for
an organism to produoe the faetor which confers resistance,
the gene for the fac~or and the mRNA for ~he factor must
be present in the cell. Thus a probe sp~cific for the
facto~ mRNA can be used to det~ct, identify, and quantitate
~; ~ 30 the organism~ whi~h are producing the factor by utilizing
nucleic acid hybridiza~ion methods.
.~ ~
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-9~3-
The above examples of the use of nucleic acid
probes specific for ~articular ~equences or populations
of sequences of ~RNA, hnRNA, snRNA or psRNA for the
purpose of detecting, identifying, and q~antitating
5 pa2ticular group~ of organisms, cells, or viruses in
cells containing such mRNA, hnRNA, snRNA or psRMA
sequences, by nueleic a~id hybridization methods, are
illustrative only, and not limiting.
The Determination of the Sensi_ivity of Microor~anisms
to Antimicroor~anism A~ents
A large number of differen~ clinical situations
require the determination of antimicroblal agent suscep-
tib~lity for a variety of differen~ bacter~ a and anti-
~: 15 biotics ( see "Antibiotics in Labora~ory Med~ eine" by
V. Lorian, Editor, Publi~er, Williams; and Wil~ens
Bal~imore, 1980) All of these situation~ utilize a
method for detecting and quantitating specific classes
2û of microorganism~. In many of these situations useof ~he nucleic acid hybridization tests described
~; ~ earlier would greatly speed up the de~ermination of
antimi~robial agene susceptibility.
As the organ~ sm in a sample grow and divide, the
25 : a~ount of RNA in ehe culture increases . A doubling of
organlsm~ r~qult~ in a two fold increase ln the qu~nti~y
of R~A~ of differen~c type~ which is pre ent in the cul~re.
~; Thu~ organism growth c n be monitored by determining the
: quantity of RNA present in the cul~ure at different times
30 after the -qta~c of growth incuba~ion. An increase in
the amoun~c of RNA presen~ w~th time ind~cates organism
.
7~387
99_
growth. The magnitude of the increase indicates the
extent of growth. The rate of growth i~ then the extent
of growth per time period. Probes specific for, R-RNA,
t-RNA, psR-RNA, pst-RNA, certain mRNAs or psm~As,
certain snRNAs or pssnRNAs~ or hnRNAs or pshnRNAs can
be used indivldually or in combination to measure the
~rowth of organisms since the quantity of each of these
RNAs in a culture will increase as the organism~ grow.
A culture of specific category of organisms grown
in the presence of an agent or agents which completely
inh~bit growth will not shown an increa~e in RNA with
time, whil~ cultures which are partially inhibited by
such age~t will show a lower ra~e of RNA accu~ulation.
A cu~ture whi~h i~ no~ inhibieed will show ~he sæ~e rate
`~ 15 of RNA increase as ~he control culture which does noe
~:~ contain the agent.
~ One example of thi~ is in de~ermining the suscep~i-
: bility of Mycobacteria tuberculocipresen~ in a clinical
;~ sputum sample. The first step in diagnosing such a
sample is to prepare a direc~ s~ear of the spu~um
for staining ~n order to detec~ acid-f2-~t bac~lli.
~:~ It i.~ esti~atet that it r~quire~ at least 104 - 105
M. tuberculosls organism~ per ml of sputum to yield
a positive direct smear. However, onIy 10 to 100 of
the~e organisms are recoverabl~ by growth culture methods.
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If ~he sputum specimen shows a positive smear,
the specimen is then treated to kill all bacterial
except Mycobacteria, and a dilution of the treated
specimen i~ plated on aBar medium containing anti-
microbial agent and on control agar which does not
contain the agent. Viable individual bacteria will
from colonies on the control agar while growth will
be inhibited on ehe agar with the appropriate an~i-
microbial agent. The ration of the number~ on the
control agar to those on the agent treated agar is then
a maeasure of the e~fectiven-s~ of the antimicrobial
agent.
A small colony will contain at lea-qt Io6 bacteria.
This meanR that at least 20 div~sion are needed to
form a colony from one bacteria and each division will
take at least lX hour~, for a total of 240 hour-~ or 10
day , at a minimum. In ~os~ cases ~t ta~e~ 2 - 4 times
thi~ long (3 to 6 week~) for colonie eo appear.
A methot describ d earlier for ~ , would
greatly decrease the ~im~ needed for deter~ining an~i-
microbial agent su~ceptibiliey. A probe ~peeific only
for R-RNA fro~ ~ember~ of the genus M~cobacterium could
- be used in such a te~t. Such a probe woult allow quanti-
tation and a detection sensitivity equal to that described
~5 earlier for L~ionella. A nucleic acid hybridization
: test u~ing ~he aecelera~ed hybritization rate conditions
and the ex~e s probe mode of hybridization would easily
allow ~he detec~ion of abou~ 200 Mycobac~eria cells. A
: step woult be atded ~o en~ure the di~ruption of ehe
~` 30 Mycob:aceeria celIs so tha~ ~he R~RNA would be free to
hybridize. ~ycobac~eria do not readily lyse in the
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1.~ 789
-:LOl-
presence of enzyme-detergent solutionq.
As mentioned above, a minim~m po~itive sputum
specimen ( as determined by acid staining) contains
about 10 to 10 ~y~ cells per ml and these
10 to 102 cells can ~e detected as colony forming units.
For drug susceptibility studies on agar, enough ~yco-
bacteria are added to the control and experimental agar
surfaces to ensure that 40 to 50 colonies will appear
: on the control agar where no antimicrobial age~ is
present. If such a practice is followed when usi-lg a
nucleic acid hybridization assay thi~ means that the
culture is star~ed with about 50 MYcobacteria and it
will the~ take about 3 - 4 cell d~visions or about 2 - 3
tay~ in order to obtain a detectable level of cells.
If any ~ignificant inhibition of growth by t~e agent
ha occurred the control will be posiei~e and the culture
: ~ containing agent will be negative. It is clear ~hat
the use of ~he highly sensitive nucleic acid hybridization
~ ~ me~hod can greatly reduee the tim~ needed to determine
; ~ 2a susceptibility by 5 to 10 fold.
The abo~e is ~ust one example of the use~ of nucleie
ac~d hybridizat~on te~ts such a~ those deseribed for
E~ for de~erming antimicrobial agent sensitivities.
The sensltivity of any microorganlsm can be determined
by utilizing a co~bination o~ ~he s~a~dard growth method-
: :ology an~ an assay for microorganim~ based on nuclei~
. acid hybritization. In addition, in many case~ the
spe~ificlty and sen~tivity of the nucleic acit hybridi-
~: : zation test~o~ ~ croorganisms allow the determination
~ ~ 30 of antibiotic sen~iel~ity of specific organis~ even in
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the pre~ence of a large excess of other microorganisms
or eukaryotic cells.
It i obvlouY th~t the ~ame approach can be used
to determine the presence o~ antimieroarganism activity
in blood, urine, other body fluids and ~ssues and
other samples. In this case my nucle~ c acid hybridizat~ on
procedure can be used to monitor and quantitate the effect
of the bLood, ur~n~ 9 or other sample on the grow~h of a
specific group of microorganismS which are put ipeo
contact with said blood, uring or other samples under
conditions where growth oc~ur~ if anti~icrobial activity
is not present.
The overall rate of pro~ein synthe~i in a c211
: 15 ic determined by the number of ribo~ome~ per cell. The
rate of t-RNA synthe~is is aLso related to the ~umber
of ribosomes per cell. Inhibition of protein synthesis
: in a cell results ~n the cessation of R-RNA synthesis by
: the cells. Indeed, stopplng cell growth by any means
results ~n th~ ce~at~on of R-RNA synthe is and slowing
cell growth re~ult~ in a slowing down of R-RNA synthesis.
~; The newly s~n~hesized R-RNA molecule is larger ~chan
the suss of ~che mature R-RNA ~ubunits prese~t in the ribosome.
25 For example the R-RMA of E. coli is -~yntheslzed as a pre^
curqor molecule 6000 baseY long. The precus~or molecule
then proces~ed to yield the R-RNA ~ubuslits (totaling
about 4500 b~ses3 w~ieh are then incorporated into
.~ r~bo o~e and "extra" or pr~cursor speci fic R-RNA
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-103-
(p~ R-R~A) sequenceq which are eventually degraded
by the cell.
R-RNA i~ not ~ynt~esized in non-growing cell~ and
therefore no precursor specific R-RNA sequences are
present in ~hese cell~. In ~his case, large numbers
of R-RNA molecule3 are present ln the cell but no
pq R-RNA sequence~ are pre.~ent.
In a ~lowly growing cell a s~all amount of R-R~A
precur~or i synthesized ant a small amount of p~R-RNA
is present.
In a rapidly growing oell a large amount of R-RNA
precur~or i~ -~ynthesized and several thousand psR-RNA
sequenees are presen~.
The ab ence of psR-RNA in a cell signal that the
cell is not growing. The ratio of R-RNA to psR-RNA in
a eell is an indication of the growth rate of a ~ell.
Antimicrob~l agents inhibit cell growth. Cells
which are not growth inhib~ted by the agene will contain
large amou~t~ of psR-RNA. In cells whioh are only
:: 20 partlally growt~ inhibited the psR-RN~ will be present
~;~ in a loer a~ount. The ratio of R-RNA to psR-RNA will
give a ~ea~ure of the degree of inh~b~tion.
A nucleic acid probe specific for the psR-RNA
~5 sequences o~ a par~icular group of microorganisms
can be used in a nucleic acid hybridization eest to
determlne and quantitate the pr~sen~e or absenca of
: psR-RNA in tho3e microorganis~ when the organisms
are growrn in the Rre ence and absence of a particular
3~ anti~icroorganis~'i~ent or a group of such agents. This
ca~ be done even in the presence of large number3 of
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~ 7~3~7
-10~
or~anisms which are not related ~o the microorgani m
group of intere~t.
It is obviou3 that this nucelci acid hybridization
method can also be used to determine the presence of
substance~ with antimicroorganism aceivi~y in blood,
urine, other body ~luids and tissue~, and other samples.
This method of determining growth of cell.~ can be
used tO determine the ~tate of growth of any cell which
synthesizes ~-RMA. The abo~e exa~ple ~ only one of
many us~d for such a method. A method based on u~ing a
~: probe specific for the p~t-RNA sequence~ of a particular
group of organisms or cellq can al~o be used to deter~ine
- the state o~ growth o~ those organ~sm~ or cells.
A method baset on u~ilizng probe specific for
cer~cain mRNAs, psmRNAs, hnRNA~, pshnRNA, snRNAs,
or pssnRNAs, which are abundanc in rapidlv growing
organisms or cells but absent, or pre~ent in low amount,
in non-growing or slow-growing cells can also be used
to determine the statP of growth o~ the~e organis~s
or cells. For example, the mRNA for a pEotein, ~NA
polyerase, i pre~ent in abundance, several hundred
copies per cell, in rapidly growing cell~. In non growing
~ cells cery little ~NA iq ~ynthe~ zed and little ~RNA
: i~ pres~nt.
: 25 A method baset o~ u~ilizlng probe speciflc ~or
er~ain ~iru~ m~NA~ or psmRNAs which are abundant when
~aid viru3 i~ rapidly gowing in a cell and ab~en~ when
the viru~ i pr~3ent in a eell bu not growing, eab also
- :~ be ~et ~o deter~ina~he ~tate of growth of viru~es in
cell
:
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-105
Thu~ in qituation9 where member~ of a particular
category of organi~m~ are known to be present in a
sample it iq po~sible to use a ~lngle probe to determine
the growth state of said organis~s. For example if no
5 p~ R-RNA can be detectet in the organisms, ~hey are in
a non-growing state. If psR-RNA is detected in ~he
organisms but in low amount relative to the number of
organisms preRent, the organisxnc ar@ growinE~ 910uly.
If large a~nounts of psRNA are detected, relati~re tO
10 th~ number of organisms present, the organismx are
grow~ng rapidly.
Another approach to determinlng the s~a~e of growth
of a particular ~rganis~n or class of orgar~isms relies in
utilizing two probeq, each of which ~ill hybridiæe only
to RNA from a particular category ~f organis~s one probe
is ~peciflc for a ~table RNA (R~RNA or t-RNA) which ~NA
~: is present in said organi~ms in roughly ehe same amount
Ln non-growing organisms or cells and rap~dly growlng
: organism~ or cells; the other probe is specific for a
20 particular mRNA, psmRNA, pst-RNA, pssnRNA, hnRNA,
p~hnRNA or psR-RNA ceqllence or sequences which RN~ is
p~eseslt in abundance in rap~dly growing cells or
organis~ns, absent or present ln low a~ount in non-growing
organism~ or cells. Thes0 probe~ are ueilized to detect,
25 iden~i:Ey, and quan ~ta~:e the amount~ present in ~he sample
of the ~ each i ~pecific for . The rat~ o of the amounts
: of the3e R~A~ i~ an indication of the growth s~ate of the
organi3m~ or cell3.
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~ .~78987
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A specific example of this ~nvolvec the u~e oftwo probes, one speclfic for the R-RNA of members of
& spec~fic category of organisms or cell~, and the other
specific for the p~R-RNA of the ame category of organisms
or cells, in order to detect, identify, and quantitate
the R-RNA and p~R-RNA present in a s~mple. The ratio
of the amount o p~RNA to R-RNA present in the sample
is an indicator of the state of grow~h of the organism
or cells. In rapidly gorwing cQll~ there are s~veral
thousand copies of psR-RNA and the psR-RNA/R~RNA ratio
is at a maximum. In slowly growing cell~ a relatively
small amount of psR-RNA i9 presenc and the psR-RNA/
R-RNA ratio $s m~ h lower. In non-grow~ng c~lls psR-RNA
shouid be absent and the p~R-RNA/R-RNA ratio is at a
15 minirrrum.
This same two probe ~ethod can bs u~ed with a variety
of different combina~ions o the probes mentioned abov2
and can be done in the presence of orgarlis~s or cell~
which are not members of the said specific category
20 de~ec~cet by the probe.
An obviou~ application of the methods described here
to deter~ine th~ state of grow~ of specific categorîes
: of organism~ is the u~e of these m~thod~ to: deeer~i~e
: the presence of antimicrobial agen~Y in blood, urine,
25 other body fluld~ or tissues or other ~amples; determine
the sen~itivity of specific categorY es of organisms to
specific sntimicrobial agent~ or groupq of ~uch agents.
For example bacteria which are completely growth inhibited
: ~ by a particular a~n~ will have a minimu~ psR^RNA/R-RNA
~ ~ 30 raclo,
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-107- ~ ~ 7~
Detectin~, Identifyin~, and Quantitatin~ Viruses
It is often important to be able to quickly
determine whether a particular virus or group of viruses
is present in a sample. This can be done by utilizing
nucleic acid hybridization tests described herein.
The rapid nucleic acid hybridization test which
combines: a) the me~hod for rapidly making nucleic
: acid available for in solution hybridization; b) the
~ethod for greatly accelerating ~he rate of nuc~eic
acid hybridization; c) and the rapid method for aqs~ying
for the presence of hybridized probe; is dirsctly appli-
caSle to the detertion, identification and quanti~a~ion
of any group of DNA or RNA viruse~ present in a sample
by ~he use of a nucleic acid probe which is c~mplementary
to the virus group of interest.
In addition, ~uch a viru3 assay mcthod could be
,u~ed to determine t~e effectiveneQs of particular anti-
viral agent and to determine the presence of antiviral
activity in blood, urine and other s~mples.
, .
Meehod for Detecting Microorganism Infections
by Exa~inin~ o~ Or~anism's Pha~cy~ic Cells
~ The extremely high sensitivi~y an~ specificity of
: detect~on characterizing the nucleic acid hybridization
~ tes~t.~ speci~lc for R-RNA which ha~e been described above,
.~ 25 permits of a simple solution to the problem of obtaining
. .
an appropriate clinical specimen for microorganism
: : : diagnoci.~. A simple blood test sample which eontain~
the white blood ~ell (hereinafter referred to as WBC)
fraction will suffice in a large number of cases.
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.-108-
One manner of using this WBC approach i~ to first
hybridize the '~BC sample with a marked probe which will
hybridize to R-~NA from any member of the group of all
bacterial but doe~ not hybridize tQ R-RNA fro~ any other
source. Such a probe ser~e~ a~ a general screening device
for any ba-teria. Sample~ which are positlve for
bacterial R-RNA are then aqsa~ed with a hierarchy of
other probes in order to further identify ~he bacteria
which s present. For example, a probe whi~h h~bridizes
to R-RNA from any member of the Famlle Enterbacter but
not to R-RNA from any other source can be used to detect
or rule out. Eneerbac~cr bacteria while a probe specific
only for anaerobic R-RNA woult be used to detect anaerobes.
The above illu~tration is just one of may possible
way~ of using the WBC~ a~ the prim3ry cl$nical sample
for the quick diagnosis of microorganis~ infections
by nucleic acid hybridization. For example, d~pending
on the clinical symptom~ of t~e patelnt, different
co~bination~ of probes would be used in order to ob~ain
a diagnosi~.
~. ~
~ 789~7
-109-
~ he publicat~ons listed below are of interest
in connection with ~arious aspects of the invention
and are incorporated herein as part of the disclosure.
1. Repeated Sequences in DNA
R. J. Britten and D.E. Kohne, Science (196~) 161 p 529
2. Kinetics of Renaturation of DNA
J. G. We~mur and N. Davidson, J. Mol. Biol.
(1968) 31 p. 349
3. Hydroxyapatite Techni ~ s for Nuclelc A~ld Rea~K~iati~n
D.E. Kohne and R.J. Britten, in Procedures in Nucleic
Acid Research (1971), eds Cantoni and Davies, Harper
and Row Yol 2, p 500
4. Hybrid$zaeion of Denatured RNA and S~all Fragme~ts
Transferred eo Nitrocellulo~e
: P.s. Thoma~, Proe. Natl. Acad. Sci. USA ~1980)
77 p 5201
5. DNA-DNA IHybridization on Nitrocellulose Filter~:
General Corl~ideration~ and Non-Ideal Xinetics
R. Flav~ll et al., Wur. J. Bioche~. (1974)
47 P 535
6. Assay of DNA~A Hyrbits by S Nuclease Digestion
and Ad~orption to DEAE-Cellul~;e Filtcrs
I. Maxwell et al., Nucleic Ac~ds Research (1978)
5 p 2033
7. Molecular Cloni.ng: A ~aboratory Manual
~: T. Mania~i~ et al., Colt Spring Harbor Publication ~1982)
: ~ 8. Efficien~ Tran~cr~ptio~ of RNA ln o DNA by Avian
Sarooma virus Poly~erase
J. Taylor et al.~ B~ochemica et Biophy-~. Acta (1976)
442 p 32~: :
~ 9. U~e of Specif~c Radioactive Probes to Stu~y Trans-
`~ ~ cription and ~epLication of the I~fluenze Virus Genome
J. Taylor~e~ al., J. Virology (1977) 2} t2. p 530
:::: `:
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-110-
10. Virus Detection by Nucleic Acid Hybridization:
Examinat~on of Normal and ALs Tissue for the
Presence of Poliovirus
D Kohne ee al., Journal of Gene~al Virolo~y (1981)
11. Leukemogensis by Bovine Leu~emia Virus
R. Kettm~nn et al., Proc. Natl. Acad. Sci. USA
(1982) 79 ~8~ 5-2469
12. Prenatal Diagnoqi of a Thalassemia: Clinical
Application of Molecular Hybridization
Y. Kan et al., New England Journal of Mediclne
(1976~ l p 1165-1167
.13. Gene Deletions in a Thalassemia Pro~e that the
5 ~ ~OCU3 i~ Funtional
L. Pressl~y et al. 9 Proc. Natl. Acad, Sci. USA
(1980) 77 ~6 p 3586-3589
14. U3e of Synthetic Oligonucleotide as Hybridiza~ion
P~obes. S.V. Suggs et al., Proc. Natl. Acad. Sci.
USA (1981) 78 p 6613
-:~ 15. .Identifica~ion of Enterotoxigenie E. coli by Colony Hybridization Using 3 Enterotoxin ~~ne Probes
S.~. Mosely el atl., J. of Infect. Diseases (1982)
: 145 ~6 p 863
: 16. DNA Reas~ociation _n thé Taxonomy o' Enteric Bacteria. ~ D. Brenner, Int. J. Systema~ic Bacteriology (1973)
23 ~4 p 29~-307
~ 1?. Compara~ive ~tudy of Ribosomal RNA Cis~rons in
I Enterobacteria and Myxoba ter~a
~: R. Moore et al., J. Bacteriology (1967)
9~ p 106~ 7~
~ 18. R~bosc~al RNA Si~ilarieie~ in the Classification
:: ~ of Rhodococcu~ and Related Taxa
. M. ~ordarsk~ et al., J. General Microb~ology tl980)
: 118 p. 313-31~r
".
19. Re~ention o'~on Nucleotide Sequeno~s in the
~: Ribo~omal R~A DNA of Eukaryote~ and Some of their
Physleal Characteristic~
J. Sinclair et al., Biochemistry (1971) 10 p 2761
. ~
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-.
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20. Homologies A~ong Ribosomal RNA and Messenger
RNA ~ene3 in Chloroplasts, Mitochondria and
E. coli
~ert et al;, Molecular and General Genetic~
(1980 179 p ~ 545
21. Heterogeneity of the Conserved Ribosomal RNA
Sequenceq of Bacillus subtili~
R Doe e al., J. BacterioTogy (1966)
22. Isolation and Characteriza~ion of Bacteria~
Ribosomal RNA Cistrons
D. Kohne, 3iophy~ical Journal (1968)
8 ~1~ p 1104-1118
23. Taxonomic Relations Between Archaebacteria
Including 6 Novel Genera Examined by CroRs
Hybridization of DNAs and 16S R-RNAs
J. Tu et al., J. Mol. Evol. (1982) 18 p 109
24. R-RNA Cistron Hbmologie Among ~YDoh~robiu~ 2nd varicuS O~r
Bacter~, R. M~ore; C~an J. ~ g77) ~3 p 478
r ~ ~"25. Conservation of Transfer RNA and 5S RNA Cistrons
in Enterobacteriaceae
D.J. Brenner e~ al., J. Bactcrlology Yol 129
~3 (Mar 1977) p I~35
26. Seqeunce Homology of Mitochondrial Leucul-tNA
C~tron in Different Organi~
S, Jako~cic ~t al., Biochemistry Vol. 14 tlO
(May 20, 19753, p. 2037
~: 27. Synthe~ic Deoxyoligonucleotide~ a Çeneral Probeq
: for Chloropla-ct t-RNA Gene.
J.A. Nic~oloff and R.B. Hallick, Nucleci Acids
~ Research, Vol. lQ ~24 (1982) p 8l9l-82lo
:~ 28. Antibiotic~ in Laboratory Medicine
~: V. Lorian ed, Willia~s and Wilken~ (Baltimore/London)
: 1980
29. Diagnostic Microbiology
Finegold and Martin, Edi~or~, C.V. Mosby Co.
(St. Louis) 1982
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~ ~789~7
-112-
30. spotbloe: A Hybridization Assay or Specific DNA
Sequence~ ln Multiple Sample~
M. Cunningham, Analytical Bio~hemistry
Vol. 128 (1983) p. 415
31. (29) Analy~is of Repeating DNA Sequences by
Reaqsoc~ation
R. Britten et al., in: Method~ in Enzumology
XXIX, page 3~, Eds. Grossman and Moldave,
Academic Pre~s, New Yor~ (19743
32. Studiec on Nucleic Acid Reassociation Kine~lcs
Retarded Rate of Hybridiation of RNA with Excess
DNA
G. Galau et al., P-oc. Natl. Acad. Sci. USA
Vol. 74 ~ T4) p 2306
33. Acceleration of DNA Renaturation Ra~e~
3. Wet~ur, Biopolymer~ VoL. 14 (1975) p 2517
34. Room Temperature Methot for Increasing ehe Rate
of DNA Reassociation by Many Thou.~andfold: The
Phenol Em~lsion Reassociation Teckniqu~
D. Kohne et al., Biochemistry Vol. 16 ~24 (1977)
35. Molecular Biology
D. Freifelder, Scienc~ Book International (Boston)
~ Van Nostrand Reinhold Co. (New York) 1983
: 36. Gene Expre~3ion 2
. ~ B. Lewin, J. Wiley ~ Sons, Wiley-In~erscience
Publlcatlon (1980) New York
:: ~
37. Gene Expres~i~n 1
:~ ~ B. Lewin, J. Wiley ~ Son~, Wiley-Interscience
Publicat~on ~1974) New York
.
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As used in the specification and claims the following
terms are definited as follows:
DEFINITION OF TER~lS
base (see n~cleo~ide)
base pair mis~atches
(see i~Derfectly
ccmDlementary base
sequence)
base seqeunee, (nucleotide A DNA or RNA lecule ccnsisting
10 sequence or ge~e sequence of mLl~iple bases.
or ~olynucleotide
sequence or single
strand nuclei~ asid
sequence)
.
15 complementary base pa~rs Certain of the base~ have a chemical
affinity for each other and pa;r
toge~her, or are comDle~gneary to
: one another. The coFplementary
: ~ase pairs are A:T nd G:C in
O DNA and A:U in RNA.
` complemEntary strand~ or Perfectly co~plYmen ~ nucleic
`- ccmplementary ba~e acid lecules are nucIeic acid
sequences moIecule ~ in which each:base in
one m~lecule is paired:with its
c ~ lementary base in ehe other
scrand, to form a stable helical
dbuble strand mDlecule. The
indiYidhal strands are termed
: complementary strands.:
30: criterion Mbs~ preeiesely defined as ehe
:~ difference between the te~perature
of nelting of the double strand
: nu~leic æ id and the temperature
at which hybridi~ation is done.
35~ The melting temperature of a
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DEFINITION OF T~FUIS (Cont'd)
crieericn, conei~ued. double strant n~cleic acid is
determnr.ed primarily by the sal~
concentra~icn of the solution.
The criterion deter¢ines the deg~ee
of co~plementæ ity ~eeded for two
single stranls to form a seable
double strand molecules. The
criteri~n can ~e described as
highly stringent, or no~ very
stringent. A highly stringent
criterion requlres that ~wo
interacting com~lem~n~ary sequences
be highly complementary in sequence
lS in order to fon~ a stable double
strand molecule. A poorly strLngent
criterion is one which all~w~ rela-
tively disslmil3r compl:mentary
strands to interacr znd form a
double strand molecul~. Hign
stringency allow~ the p~esence of
only a small ~raction of b~se pair
` -~ mQsmstches m a doubIe str2~d ~ole-
cule. A poorly stringent criterion
` ~ 25 allows a much Iarger ~raction of
base pair mismatches in the hybridi-
zation pr~duet.
e bont betw~ the paired bases in
denæ~ed or d~ssoclated a do~ble g~and rlucleic acid molecule
30 ~ ~lelc æld : ~ : can be broken, resultinz ~n ~o
single s~r~d m~lecul~s, ~which
then dif~use ~way fran each oth.er
d~le 8traSlt; ~n~cle~c acid As it is fa~t in eh.e cell, ~ost DNA
is in the d~le st~d staee. The :~
35 ~ :DNA is :made up o two:~DNA ~leculesor ~ strands ~und~ h~lically OEo~d
eac~ other. : The: bases fa~e i~ard ::
:i and each bas~ is~specifically
bondet eo a canç~lementary base in~
4:0: ~ the ot~ir strand. For exa~le, a
A in or~!e s~rant is always paired
h :a T~ ~ ~he other strar.d, ~ile
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DEFINITION OF TE ~ S (Cont'd)
do~ble strand nucleic a G in one s~Iand is paired with a
acid, con~nued. C in the other strand. In a
bacterial cell th~ double strand
molecule is abcu~ 5 x 106 base
pairs long. Each of the bases
in one strand of this ~olecule
is paired with its base ccmDle~.ent
in the other strand. The base
se~uences of the individual double
st~and molecules are termed
comple~entary strands.
hybri~;~ation ~see
nucleic and hybridization)
; i~perfectly cc~plementary Stable double strand ~Dlecules can
15 base sequences (base pair ~e form.ed between rwo strands where
mismatches) a fraction of the bases in the one
strand are paired with a nan-
complement æ y base in the other
strand.
20 marke~ probe or ~arked Single strand nucleic aoid ~Dlecules
s ~ ce which are used to deeect the presence
of o~her nucleic acids by the process
of nucleic acid hybridization. The
probe molecules are ~ark~d so that
they can be specifically detected.
This is done by incorporating a
; specific marker ~olecule into ~he
nNcleic acid or by attaching a
;~ specifie marker to ~he nucleic
acid. The st effec~ive probes
~ ~ are ~arked, single s~r~nd sequences,-~ which cannot self hybridize but canhybrid~ze only if the nucleic acid
to be~detected is present. A large
; ~ 35 number of differEnt ~ar~ers are
available. These include radio-
active and fluorescent ~olecules.
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6-
DEFINITION OF TERMS (Cont'd)
nucleic acid hybridizatiqn The bond ~etween the cwo strands
cr hybridization (reassociation, of a double strar.d molecule c~n
or renarurati~n) be br~ken and the tw~ single
. strands c~n ~e comDletely
sep~ated frcm each ot~er. ~nder
ehe proper cond ticns the corDle-
meneary single st~nts c3n collide,
~ecogni7 each other and refo~3
the double str~nd helical m~lecule.
This process of formaticn of double
strand molecules ~r~m comple~entary
single strand molecules is called
nucleic acid hybridization.
Nucleie acid hybridizaticn also
occurs benween partially c~mple-
~entary single s,rands of R~ and
D~A.
nucleotide, nucleotide M~st DNA consists o~ sequences of
base or base only four nitrogeneous bases:
adenine (A), thynrL~e (T), guanir~e
(G~, 2nd cytosir~ (C). Together
these bases fo~ the genetic
alphabet, and lGng ordered
sequences of ~he~ coneain, in
coded fon~, m~h of t~e i~far~aticn
present in genes.
Mbs~ RNA also c~nsists of sequences
: of only four bases. However, in R~A,~ ~: 30 ~ th~e is replaoed by ulidine (U).
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-117-
DEFINIIION OF TEgMS (Ccnt'd)
reassociaticn (see ~cleic acid hybridizaticn)
rena~uration (see nu~leic acld hybr~di7ation)
ribos al R~A or R-RNA The R~A which is presenc Ln ribosomes.
Virtually all ~ibosomes c taLn 3
s ~ le strand RNA suDunits: one
large, one medium-sized, and one
small.
riboscme A cellular par~icle (containing RNA
: 10 and protein) necessary for protein
synthesis. All life forms except
viruses contain riboscmes.
R-RNA DNA or The base sequence ~n the DNA which
codes for ribosomal RNA. Each R-~NA
R-RNA ge~e subunit is co~ed for by a separate
.
R-gM~ probe A m æ ~et nucleic acid sequence which
ls cc~pleIent~ry to R-R~A ~nd there-
: fore will hybridi2e with R-RNA to
form a stable double strand molecule.
mgNA Each individual niU~A is a direct gene
: product containing ~he information
necess~ry to specify a p rticular
prote~n. The ma~hinery o the cell
tr2nslate~ ~he sequence of the ~RNA
. into a specific pro~ein. Many
tifferent ~RNAs exi~t ~n each cell.
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DEFI~ITION OF IERMS (Cont'd)
hnRNA A c ~ lex class of ~NA sequences
presen~ in the nucleu~ of eukaryotie
cells which includes precursor ~RNA
~olecule~. Most h~ R sequ~nc~s
never lea~e the nucleus. The
func~ion af mos~ of chese ~Dleclles
in ~awn.
snRNA A c13ss of relaeively stable s~all
nuclear RNA lecules which æ e
present pr ~ ily in the nucleus of
~: eukaryotic cells in large n ~ rs.
precursor ~ M~DIY RNA mD~eculec in both prokarvotes
: ~nd eukaryote~ are synehesi7ed as part
:~ I5 of a large gNA m~lecules which i5 ~hen
processed to ~ eld ~ature RNA ~Dlecules
- of v æ icus types 2nd o~her ~ ller
sequences which are apparently
tiscarded.
:, :
: ~ 20 pre~ursor specific ~NA T~ RNA sequences present in ~re~rsor
` ~ ~p ~NA). ~NA, t-~NA, ~-RNA, snRNA, an~ h¢~YA
w~ich are t ?resent in the mat~;e
: R~ RNAI n~, snRNAI and ~NA
: moleo~les.
; 25 eher=~l stabili~y of The thermal seability or melting
: double strant nucleic population of d~uble strand mDlecules
acid mDlecules ~ha~ been converted to the single
scr~hd for~.
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DEF~NIrION OF T~MS (Cont'd)
restric~ion ~ eq Compcnents of the restricticn-
~odificatio~ cellul æ defense
system agains~ foreign nucleic
acids. These enzymes cue
unmodified (e.g., methylated)
dcuble-stranded DNA at specific
sequences ~hich exhibit ~ofold
sym~etry about a poine.
tr~nsfer RNA ~t-RNA~ During protein synthesis individual
zminD acids are aligned ~n the proper
order by v æ ious specific ad~ptor
nolecules or e-KN~ ~ole ~ es. Each
amino acid i osdered by a different
t-RNA specie~.
le the invention has been described ~nd illustrated in detail,
it will be apparent to those sXille~ in the art that various
ch~nges, equivalents and alternat~ve~ may be resorted to without
dep æ tir~ fro~ the spIrit of the i~vention, and all of sush
2~ ~ es, eou~valents 2nd alternative~ are cont~mplated as ~ay
come within t~e scope of th~ appended claims ~nd equivalents
~reof.
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