Note: Descriptions are shown in the official language in which they were submitted.
i~ ~ 31~7~
CROSS-REFERENCES TO RELATED APPLICATIONS
This application is related to Canadian
Application S.N. 530,235 filed February 20, 1987.
~CKGROUND OF THE INVENTI~N
Field o~ the Inventlon
The present application relates to the
detection and identification of microorganisms and the
detection and identification of particular prokaryotic or
eukaryotic D~A sources in a nucleic acid containing test
sample.
Still further, the present invention relates to
a method for the lysis of whole cells.
Background Information
A. The Detection of Microor~anisms
The identification of species of microorqanisms
in a sample containing a mixture of microorganisms, by
immobilizing the DNA from the sample and subjecting it to
hybridization with a labelled specimen of species -
speci~ic DNA from a known microorganism and observing
whether hybridization occurs between the immobilized DNA
and the labelled specimen, has been disclosed in PCT
patent application No. PCT/US83/01029.
The most efficient and sensitive method of
detection of nucleic acids such as D~A after
hybridization requires radioactively labeled DNA. The
use of autoradiography and enzymes prolongs the assay
time and reauires experiellced technicians.
U.S.P. 4,353,535 to Falkow et al describe
infectious disease diagnosis using labeled nucleotide
probes complementary to nucleic acid coding for a
c~lara(~eri.~itlc pa thogen pr oduct.
~3~4794
B. The Detection of Specific Eukaryotic Sequences
The identification of specific sequence
alteration in an eukaryotic nucleic acid sample by
immobllizing the DNA from the sample and subjecting it to
hybridization with a labeled oligonucleotide and
observing whether hybridization occurs between the
immobilized DNA and the labeled probe, has been described-
It is known that the expression of a specificgène determines the physical condition of a human being.
Adult hemoglobin is a tetrameric association o~ two alpha
and two beta subunits. During embryonic and etal life,
the alpha chains are associated with, successively, gamma
and delta chains before the adult beta form predominates.
The genes for alpha hemoglobin are located on human
chromosome number 16 and tlle genes ~or gamma, delta and
beta h~moglobin are tandemly linked on human chromosome
ll. Hemoglobinopathies are heritable diseases that are
the result of alterations in the structure of one or more
of the hemoglobin genes. Many of the mutations have been
characterized in considerable molecular detail and can
range fronl single bas~ pair changes to wholesale deletion
of a gene family. For e~ample, a change in the
beta-globin gene coding sequence rom GAG to GTG at the
sixth amino acid position produces sickle-beta-globin and
a homozygate can have a disease known as slckle cell
anemia. Similarly deletion of particular sequences from
alpha-globin or beta-globin genes can cause thalassemias.
A recent survey, The ~ew Genetics and Clinical Practice,
D. J. Weatherall, The Nuffield Provincial ~ospitals
Trust, (1982), chapter 2 describes the frequency and
clinical spectrum of genetic diseases.
13~79~
Problems associated with genetic defects can be
diagnosed by nucleic acid sequence information. The
easiest way ~o detect such sequence information is to use
the method of hybridization with a specific probe of a
known sequence.
U.S.P. ~,395,48~ to Wilson et al describe a
method for the direct analysis of sickle cell anemia
using a restriction endonuclease assay.
Edward M. I~lbin and Yuet Wai Kan, "A Simple
Sensi~ive Prenatal Test for l~ydrops Fetalis Caused By
~-Thalassaemia", The Lancet, January 12, 19~5, pp. 75-77
describes a do-t blot analysis to differentiate between
the genotypes of homozygous alpha-thalassemia and those
o~ the haemoglobin-~l disease and alpha-thalassemia trait.
The most efficient and sensitive method of
detection of nucleic acids, such as VNA, after
hybridization requires radioactively labelled DNA. The
use of autoradiography and enzymes prolongs the assay
time and requires experienced technicians.
Recently, a non-radioactive method of labelling
DNA was descrihed by Ward et al, European Patent
ApplicatioJl 63,~79. Ward et al, use the method of nick
translatioll to introduce biotinylated U (uracil) residues
into DNA, replacing T (thymine). The biotin residue is
then assayed wi~h antibiotin antibody or an avidin-
containing system. The detection in this case is quicker
than autoradiography, but the nick translatioll method
requires highly skilled personnel. Moreover,
biotinylation using biotinylated UTP (uridine
triphosphate) works only for thymine-containing
polynucleotides. The use of other nucleoside
triphosphates is very difficult because the chemical
derivatization of A (adenine) or G (guanine) or C
(cytosine) (containing -N~12) with biotin requires the
~s~ills oE trained or~Janic chemi.s~s.
~L3~79~
C. Effect of Nonionic Polymers on liybridization
It has been shown by Wetm~lr (Biopolymers, 14,
2517-2524, (1974)) that anionic polymers such as dextran
sul~ate increase the rate of DNA-DNA hybridization in
solu~ion. It has also been shown that the heterogeneous
pllase hybridization rate can also be increased by dextran
sulfate. ~.S. Patent 4,302,204 discloses the effect of
charged polysaccharides on the rate of nucleic acid
hybridization.
Recently Amasino (_nalytical Biochemistry, l_ ,
304-307 (1986)) has shown that a neutral polymer li~e
polyethylene glycol can increase the rate of DNA-R~A
hybridizatioll more than dextran sulfate under optimum
conditions. Although polyethylene glycol (PEG) has been
used before in the hybridization medium (e.g., ~enz and
Xurz, Nucleic Acids ~es., 12, 3435-3444 (1984)), no
advantage was predicted over the use o~ dextran sulfate.
Applicants have observed, to their surprise, that in the
presence of 10% polyethylene glycol hybridization can be
virtually finished in 15 minutes. Although Amasino
has shown PEG is better than dextran sulfate as an
accelerator, his labeled probe was single stranded ~NA.
In the case of the present invention, the labeled
material is whole gènomic sample DNA where ~oth
complementary strands are present in solution. When a
polymer accelerates the rate of hybridization it should
also help the reannea].i.ng in solution, and hence should
reduce the efficiency of hybridization, instead of
increasing it.
D. Cell Lysi.s
The present invention also provides a method
for the efficient lysis of whole cells such that their
DN~ is released and made available for photochemical
labeling. While eukaryotic cells derived from
nlulticell~lar animals are easily lysed under relatively
7 ~ ~L
mild conditions, single cell eukaryot~s and prokaryotes,
especially Gram positive prokaryotes, are more difficult
to lyse due to the complicated chemical nature and extent
o~ cross-lin~ing of their cell ~alls. Methods do exist
for effici~ntly lysin~ these refracto~y organisms, either
by chemical--enzymatic or physica] means, hut these
rnethods are ofterl complicated, time-consumlng and
inappropriate for preserving the inte~rity of DNA.
SUMMARY OF THE INVENTION
The present invention provides a rapid and
convenient method for detection of one or more micro-
organisms or nucleic acid sequences from a prokaryotic
source or an eukaryotic source in a nucleic acid-containing
test sample and also a method for a simultaneous assay for
the presence of more than one nucleic acid sequence.
The invention also involves direct labeling of the
nucleic acid in the test sample and the use of whole
chromosomal nucleic acid as the probe and/or as the test
sample.
Also the invention relates to the use of
oligonucleotides as immobilized probes.
The invention further relates to the use of
cloning vectors and their derivatives for use as
immobilized probes.
The invention also concerns the use of polyethylene
glycol as a hybridization accelerator.
Still further the invention is directed to preparing
a cell lysate by contacting a cell with alkali.
These advantages are realized in accordance with
the present invention for a method of detecting (i) one
or more microorganisms or (ii) nucleic acid sequences
from a prokaryotic source or
~31~79~
an eukaryotic source in a nueleic aeid-eontaining test
sample.
~he method involves the following:
(a) labeling the nueleie aeids, e.g., the
nueleic acids of the organisms or eells or cell debris,
in the test sample,
(b) contacting, under hybriclization
conditions, the labeled hybridizable sample nucleie acid
and one or more immobilized hybridizable (e.g.,
single-stranded) nucleie acid (e.g., DNA) probes
eomprising of (i) one or more ~nown microorganisms or
(ii) nueleie aeid sequences from eukaryotie or
prokaryotic sources, to form hybridized labeled nucleic
aeids and
(c) assaying for the hybridized nucleie aeids
by detecting the label.
The hybridizable nucleic acid can be whole
genomie nueleie aeid or a fragment thereof, e.g., an
oligonueleotide.
In step (a), the nueleie aeids ean be labeled
direetly in the test sample. In step (a), in sueh direet
labeling, eells of the test sample ean be lysed and then
a labeling reagent ean be added, whereby to label the
nueleie aeid.
The method further comprises denaturing the
labeled nucleie aeids from step (a) to form labeled
denatured nucleic acids.
Aeeording to the invention, a labeled nueleie
aeid test sample is eontaeted simultaneously with several
different types of nucleie aeid, e.g., DNA, probes for
hybridization. The nueleie aeid test sample is labeled
and hybridized with several unlabeled immobilized probes.
The positions o~ the probes are fixed, and the labeled
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probe detected after hybridization will i.ndicate that the
test sample carries a nucleic acid sequence complementary
to the corresponding probe.
Nucleic acid probes for scveral microbiological
systems or for different alleles of one or more genes can
be immobilized separately on a solid support, for
example, nitrocellulGse paper. The test sample nucleic
acids are labeled and remain in solution. The solid
material containing the immobilized probe is brought in
contact with the labeled test nucleic acid solution under
hybridization conditions. The solid material is washed
free of unhybridized nucleic acid and the label is
assayed. The presence of the label with one or more of
the probes indicates that the test sample contains
nucleic acids substantially complementary to those probes
and hence originate, for example, from an infection by
particular microbiological systems.
Labeling can be accomplished in a whole living
cell or a cell lysate, and can be non-isotopic. The
nucleic acid can be used for hybridization without
further purification.
The presen~ invention also concerns specific
lysis conditions to release nucleic acids from both gram
positive and gram negative bacteria.
The present invention also relates to a
hybridization medium which accelerates the process of
hybridization. A hybridization accelerator according to
the present invention is polyethylene glycol.
The present invention further concerns a kit
for detecting (i) one or more microorganisms or (ii)
nucleic acid sequences from a prokaryotic source or an
eukaryotic source in a nucleic acid-containing test
sample comprising
i 131~79~
(a) a support solid containing hybridiz~ble,
e.g., single-stranded, nucleic acid, e.g., DNA or an
oligonucleotide, of (i) said one or more known
microorganisms or (ii) said sequences from eukaryotic or
prokaryotic sources immobilized thereon, e.g., a strip
containing dots or spots of known microorganisms or
eukaryotes or prokaryotes,
(b) a reagent for labeling the nucleic acid of
the test sample,
(c) a reagent for releasing and denaturing
nucleic acld, e.g., DNA, in the test sample, and
(d) hybridi~ation reagents.
For chemil~minescence detection of the
hybridized nucleic acid, the kit would further comprise a
reagent for chemiluminescent detection.
In the above described kit, the reagent for
labeling is given her~inbelow in a discussion on labels.
Reagents for releasin~ and denaturing DNA
include sodium hydroxide and lysing agents such as
detergents and lysozymes.
Typical hybridization reagents includes a
mixture of sodium chloride, sodium citrate, SDS (sodium
dodecyl sulfate), bovine serum albumin, nonfat milk or
dextran sulfate and optionally formamide.
The present invention further concerns a method
of hybridization comprising contacting two complementary
single stranded nucleic acids under hybric1ization
conditions, at least one of which i~ immobilized wherein
polyèthylene glycol is added.
BRIEF DESCRIPTION OF TI~E FIGURES
... . . .. _ . _
Fig. 1 is an autoradiograph of results of
immobilization of an oligonucleotide sequence specific
for hemoglobin mutation.
~31~ 79~
Fig. 2 is a photograph of results of
hybridization with labeled genomic DNA for non
radioac-tive detection.
DETAILE~ DESCRIPTION OF TI~E INVENTION
.
The test sample in the present invention
includes body fluids, e.g., urine, blood, semen,
cerebrospinal fluid, pus, amniotic Fluid, tears, or
semisolid or fluid discharge, e.cJ., sputum, saliva, lung
aspirate, vaginal or urethal discharge, stool or solid
tissue samples, such as a biopsy or chorionic
villispecimens. Test samples also include samples
collected with swabs ~rom the skin, genitalia, or throat.
All these test samples can be used for
hybridization diagnosis after labeling the nucleie acids
of the sample. The labeling can be accomplished without
any processing of the sample. As for example, a urine
sample (suspected of bacterial infections) can be labeled
without centrifuga~ion, fil~ration or dialysis. The
cells in the samples can be lysed without any separation
step. It is surprising that a nucleic acid labeling
reaction takes place in such a eomplex mixture as a
clinical sample.
The nucleic aeid is preferably labeled by means
of photochemistry, employing a photoreactive DNA-binding
~uroeoumarin or a phenanthridine eompound to link the
nueleic aeid to a label whieh can be "read" or assayed in
eonventional manner, ineluding fluoreseenee detection.
The end p~oduct is thus a labeled nueleie acid comprising
(a) a nucleic acid component, (b) an intercalator or
other DNA-binding ligand photoehemieally linked to the
nucleie acid componen~, and (e) a label ehemieally linked
to (b).
The photoch~mical method provides more
~avoLa~le reaction conditions than the usual chemical
~3~7~
couplin~ method for biochemically sensitive substances.
The intercalator and label can first be coupled and then
photoreacted with the nucleic acid, or the n~cleic acid
can first be photoreacted with the intercalator and then
coupled to the label.
A general scheme for coupling a nucleic acid,
exemplified by double-stranded DNA, -to apply a label, is
as rollows:
Label
Photoreactive
Intercalator
Labeled Double-Stranded DNA + Photoreactive
Photoreactive Intercalator
Intercalator
~- DNA h~-~
h~ Chemically - Functionalized DNA
~ + Label
Labeled DNA
Where the hybridizable portion of the nucleic
acid is in a double stranded form, such portion is then
denatured to yield a hybridizable single stranded
portion. Alternatively, where the labeled DNA comprises
the hybridizable portion already in single stranded form,
such denaturation can be avoided if desired.
Alternatively, double stranded DNA can be labeled by the
approach of the presellt invention after hybridization has
occurred using a llybridization format which generates
double stranded VNA only in the presence of the sequence
to be detected.
To produce specific and efficient photochemical
products, i.t is desirable that the nucleic acid component
131~7~4
arld the photoreac~ive intercalator compound be allowed to
react in the dark in a specific manner.
For coupling to DNA, arninomethyl psoralen,
aminomethyl angelicin and amlno alkyl ethidium or
methidium azides are particularly useful compounds. They
bind to dou~le~stranded DNA and only the comple.~ yields
photoadduct. In the case where labeled double-stranded
DNA must be denatured in order to yield a hybridizable
single stranded region, conditions are employed so that
simultaneous interaction of two strands of DNA with a
single photoadduct is prevented. It is necessary that
the frequency of modification alon~ a hybridizable single
stranded portion of the nucleic acid not be so great as
to substantially prevent hybridization, and accordingly
there preferably will be not more than one site of
modification per 25, more usually 50, and preferably 100,
nucleotide bases. Angelicin derivatives are superior to
psoralen compounds for monoadduct formation. If a
single-stranded DNA nucleic acid is covalently attached
to some extra dou~le-stranded DNA, use of phenanthridium
and psoralen compounds is desirable since these compounds
interact specifically to double-stranded DNA in the dark.
The chemistry for the synthesis of the coupled reagents
to modify nucleic acids for labeling, described more
~ully hereinbelow, is similar for all cases.
I`he nucleic acid component can be single or
double stranded DNA or RNA or fragments thereof such as
are produced by restriction enzymes or even relatively
short oligomers.
The ~NA-binding ligands of the present
invention used to link the nucleic acid component -to the
label can be any suitable photoreactive form of known
DNA-binding ligands. Particularly preferred DNA-binding
ligands are intercalator compounds such as the
~uroco~rnarins, e.9., ange~licin ~isor!soralen) or psoralen
131~794
or derivd~ es thereof which pl~o~ochemically will react
with nucleic ~cids, e ~., 4 -aminomethyl-4,5 -dimethyl
angelicin, 4 -an~inometllyl-trioxsalen (4 aminomethyl-
4,5 ,8-trimethyl-psoralen), 3-carboxy-5- or -8-arnino-
or-hydroxy-psoralen, as well as mono- or bis-azido
aminoalkyl methidium or etllidium compounds.
Particularly useful photoreactive forms of
intercaiating agents are the azidointercalators. Their
reac~ive nitrenes are readily generated at long
wavelength ultraviolet or visible light and the nitrenes
of arylazides prefer insertion reactions over their
rearrangement products (see W}lite et al, Methods in
Enzymol., 46, 649 (1977)). Representative intercalatlng
agents include azidoacridine, ethidiurn monoazide,
ethidium diazide, ethi.dium dimer azide (Mitchell et al,
JACS, 10~, 4265 (198?) ), i-azido-7-chloroquinoline, and
2-azidofluorene. ~ specific nucleic acid binding azido
compound has been described by l:orster et al, Nuclelc
Acid Res., 13, (1935), 745. The structure of such
compound is as follows: ~
~IN ~H
_ C~ ( C l i ~ ) 3 - Il`' - ( C 112 ) 3 N 11 C ( 2 q ts /
N02
Other useful photoreactable intercalators are the
furocoumarins whicll ~orm (2+2) cycloadducts with
pyrimidine residues. ~lkylating agents can also be used
such as his-chloroetllylamines and epoxides or aziridines,
e.g., aflatoY~ins, polycyclic hydrocarbon epoxides,
mitomycin and norphillir~
NonlirnitincJ eY~amples oE intercalator compounds
l:,r usc i~ c tj-ese~ invel~ioll include acridine dyes,
' 13147~
phenanthridines, phenazlnes, furocoumarins,
phenothiazines and quinolines.
The label which is linked to the nucleic acid
component according to the present invention can be any
chemical group or residue having a detectable physical or
chemical property, i.e., labeling can be conducted by
chemical reaction or physical adsorption. The label will
bear a functional chemical group to enable it to be
chemically linked to the intercalator compound. Such
labeling materials have been well developed in the field
of immunoassays and in general most any label useful in
such methods can be applied to the present invention.
Particularly useful are enzymatically active groups, such
as enzymes (see Clin. Chem., (1976), 22, 1243), enzyme
substrates (see British Pat. Spec. 1,548,791), coenzymes
(see U.S. Patent Nos . 4,230,797 and 4,23~,565) and enzyme
inhibitors (see U.S. Patent No. 4,13~,792; fluorescers
(see Clin. Chem., (1979), 25/ 353), and chromophores
including phycobiliproteins; luminescers such as
chemiluminescers and bioluminescers (see Clin. Chem.,
(1979), 25, 512, and ibid, 1531); specifically bindable
ligands, i.e., protein binding ligallds; and res.idues
com~rising radioisotopes such as 3H, 35S, 32p, 125I, and
14C. Such labels are detected on the basis of their own
physical properties (e.g., fluorescers, chromophores and
radioisotopes) or their reactive or binding properties
(e.g., enzymes, substrates, coenzymes and inhibitors).
For example, a cofactor-labeled nucleic acid can be
detected by adding the enzyme for which the label is a
cofactor and a substrate for the enzyme. A hapten or
ligand (e.g., biotin) labeled n~cleic acid can be
detected by adding an antibody or an antibody pi~ment to
the hapten or a protein (e.g., avidin) wllich ~inds the
ligand, tagged with a detectable molecule. An antigen
can alsc~ be used as a ].abel. Such detec~able n-olecule
14
~ ~3~79~
can be some molecule with a measurable physical property
~e.g, fluorescence or absorbance~ or a participant in an
enzyme reaction (e.g., see above list). For example, one
can use an enzyme which acts upon a substrate to
generate a product with measurable physical property.
Examples o~ the latter include, but are not limi-ted to,
beta-galactosidase, alkaline phosphatase, papain and
peroxidase. For ln situ hybridization studies, ideally
the final product is water insoluble. Other labels,
e.g., dyes, will be evident to one having ordinary skill
in the art.
The label will be linked to the intercalator
compound, e.g., acridine dyes, phenanthridines,
phenazines, furGcoumarins, phenothiazines and ~uinolines,
by direct chemical linkage such as involving covalent
bonds, or by indirect linka~e such as by the
incorporation of the label in a microcapsule or liposome
which in turn is linked to the intercalator compound.
~ethods by which the label is linked to the intercalator
compounds are essentially known in the art and any
convenient method can be used to perform the present
invention.
Advantageously, the intercalator compound is
first combined with label chemically and thereafter
combined with the nucleic acid component. For example,
since biotin carries a carboxyl group, it can be combined
~ith a furocoumarin by way of amide or ester formation
withou~ interfering with the photochemical reactivity of
the ~urocoumarin or the biological activity of the
biotin, e.g.,
~31~79~
( i ) N ~ 2 ) 4 - C - O -
o
8iot in-l`l-hydroxysuccinirnicle
or ~ RNH 2
(ii) o ~N ~S O
H ~J--I_(CH2 ) 4 - C - (~--U2
3iotin-p-nitrophenyl ester
N ~ ( C~ 2 ) 4 - C - NHR
or
carbodiin ide
B iot in ~ ROH > Biot in CO OR
By way o~ examp le,
CH2Nilz ~
O~o ~ NH
(CH~ \ 4C00 4=~--N2
Arnt
aiot in nitrophellyl es~er
H
,~ ,"~7
tRNH2) CH2-NH-CO- (CH2) q
b~o
1~
1 31~79~
Other aminomethylanyelicin, psoralen and
phenanthridium derivatives can be similarly reacted, as
can phenanthridium halides and derivatives thereof such
as aminopropyl m~thidium chloride, i.~.,
2 ~ - ~ NH2 Cl
/ C~13
~ = C - ~H - C~ - CH~ 2 - NH2
(see Hertzberg et al, J. Amer. Chem. soc., 104, 313
(1982)).
Alternatively, a bifunctional reagent such as
dithiobis succinimidyl propionate or 1,4-butanediol
diglycidyl ether can be used directly to couple the
photochemically reactive molecule with the label where
the reactants have al~yl amino residues, again in a known
manner with regard to solvents, proportions and reaction
conditions. Certain bifunctional reagents, possibly
glutraldehyde may not be suitable because, while they
couple, they may modify nucleic acid and thus interfere
with the assay. Routine precautions can be taken to
prevent such difficulties.
The particular sequence used in making the
labeled nucleic acid can be varied. Thus, for example,
an amino-substituted psoralen can first be
photochemically coupled with a nucleic acid, the product
having pendant amino groups by which it can be coupled to
the label, i.e., labeling can be carried out by
photochemically reacting a DNA binding ligand with the
nucleic acid in the test sample. Alternatively, the
13~79~
psoralen can first be coupled to a la~el such as an
enzyme and then to the nucleic acid.
~ s described in pending Canadian patent application
Serial N;o. 486,781, filed July 15, 1985, the present
invention also encompasses a labeled nucleic acid
comprising (2) a nucleic acid component, (b) a nucleic
acid-bindincJ ligand photochemically linked to the nucleic
acid component, (c) a label and (d) a spacer chemically
linking (b) and (c).
~ dvantageously, the spacer includes a chain of
up to about 40 atoms, preferably about 2 to 20 atoms,
selected from the group consisting of carbon, oxygen,
nitrogen and sulfur
Such spacer may be the polyfunctional radical
of a member selected from the gxoup consisting of
peptide, hydrocarbon, polyalcohol, polyether, polyamine,
polyimine and carbohydrate, e.g., -glycyl-qlycyl-glycyl-
or other oligopeptide, carbonyl dipeptides, and
omega-amino-alkane-carbonyl radical such as -NH
(CH~)5-CO-, a spermine or spermidine radical, an alpha,
omega-alkanediamine radical such as -NH-~CH2)6-N~I or
-HN-CH2-C~2-N~, or the like. Sugar, polyethylene oxide
radicals, glyceryl, pentaerythritol, and like radicals
can also serve as the spacers.
These spacers can be directly linked to the
nucleic acid-binding ligand and/or the label or the
linkages may include a divalent radical of a coupler such
as dithiobis succinimidyl propionate, 1,4-butanediol
diglycidyl ether, a diisocyanate, carbodiimide, glyoxal,
~lutaraldehyde, or the like.
The spacer can be incorporated at any stage of
the process of making the probe.
a-b-d-c
defined hereinabove. Thus, the sequence can be any of
~he ollowitlg:
18
13~479~
a~b+d+c,
b+d+c~a,
d+c+b+a,
b-~d+a~-c, etc.
The conditions for the individual steps are
well ~nown in chemistry.
If the label is an enzyme, for example, the
product will ultimately be placed on a suitable medium
and the exten~ of catalysis will be determined. Thus, if
the enzyme is a phosphatase, the medium could contain
nitrophenyl phospllate and one would monitor the amount of
nitrophenol generated by observing the color. If the
enzyme is a beta-galactosidase, the medium can contain
o-nitro- phenyl-D-galacto-pyranoside which also will
liberate nitrophenol.
The labeled nucleic acid of the present
invention is applicable to all conventional hybridization
assay formats, and in general to any format that is
possible bas~d on formation of a hybridiæation product or
aggregate comprising the labeled nucleic acid. In
particular, the unique labeled nucleic acid of the
present invention can be used in solution and solid-phase
hybridization formats, including, in the latter case,
formats involving immobilization of either sample or
probe nucleic acids and sandwich formats.
The nucleic acid probe will comprise at least
one hybridizable, e.g., single-stranded, base sequence
substantially complementary to or homologous with the
sequence to be detected. ~lowever, such base sequence
need not be a single continuous polynucleotide segment,
but can be comprised of two or more individual segments
interrupted by nonhomologous sequences. These
nonhomologous seque~nces can ~e linear or they can be
self-complementary and form hairpin loops. In addition,
~:hc homolocJous reCJion ol the probe can be flanked at the
131~7~
3' - and 5' termini by nonhomologous sequences, such as
those comprising the DNA or ~NA or a vector into which
the homologous sequence had been inserted for
propagation. In either instance, the probe as presented
as an analytical reagent will exhibit detectable
hybridization at one or mor~ points with sample nucleic
acids of interest. Linear or circular hybridizable,
e~g., single-stranded polynucleotides can be used as the
probe element, with major or minor portions being
duplexed with a complementary polynucleotide strand or
strands, provided that the critical homologous segment or
segments are in single-stranded form and availabl~ for
hybridization with sample VNA or RNA. Useful probes
include linear or circular probes wherein the homologous
probe sequence is in essentially only single-stranded
form (see particularly, Hu and Messing, Gene, 17:271
(1982)).
The nucleic acid probe of the present invention
can be used in any conventional hybridization technique.
As improvements are made and conceptually new formats
are developed, such can be readily applied to the present
probes. Conventional hybridization formats which are
particularly useful include those wherein the sample
nucleic acids or the polynucleotide probe i5 immobilized
on a solid support (solid-phase hybridization) and those
wh~rein the polynucleotide species are all in solution
(solution hybridization).
In solid-phase hybridization formats, one of
the polynucleotide species participatill~ in hybridization
is fixed in an appropriate manner in its single-stranded
form to a solid support. Useful solid supports are well
known in the art and include those which bind nucleic
acids either coval~ntly or noncovalently. Noncovalent
supports which are generally understood to involve
hydro~ho~ic hondi.ng include naturally occurring and
1314L7~L
synthetic polymeric materials, such as nitrocellulose,
derivatized nylon and lluorinatcd polyhydrocarbons, in a
variety of forms such as filters, beads or solid sheets.
~ovalent binding supports (in the form of filters, beads
or solid sheets, just to mention a few) are also useful
and comprise materials having chemically reactive groups
or groups, such as dichlorotria2ine,
diazobenzyloxymethyl, and the like, which can be
activated for binding to polynucleotides.
It is well known that noncovalent
immobilization of an oligonucleotide is ineffective on a
solid support, for example, on nitrocellulose paper. The
present invention also describes novel methods of
oligonucleotide immobilization. This is achieved by
phosphorylation of an oligonucleotide by a polynucleotide
~inase or by ligation of the S'-phosphorylated
oligonucleotide to produce multi-oligonucleotide
molecules capable of immobilization. The conditions for
~inase and ligation reaction have been described in
standard text books, e.g., Molecular Cloning, T.
Maniatis, E.F. Fri-tsch and J. Sambrook, Cold Spring
Harbor Laboratory, (1982), pages 1-123.
A typical solid-phase hybridization technique
begins with immobilization of sample nucleic acids onto
the support in single stranded form. This initial step
essentially prevents reallnealillg of complementary strands
from the sample and can be used as a means for
concentrating sample material on the support for enhanced
detectability. The polynucleotide probe is then
contacted with the support and hybriclization detected by
measurement of the label as described herein. The sol.id
support provides a convenient means for separating
labeled probe which has hybridized to the sequence to be
detected from that which has not hybridized.
~3~47~
Another method of interest is the sandwich
hybridization technique wherein one of two mutually
exclusive frayments of the homologous sequence of the
probe is immobilized and the other is labelled. The
presence of the polynucleotide sequence of interest
results in dual hybridlzation to the imrnobilized and
labelled probe segments. See ~ethods in ~nzymology,
65:468 (1980) and Gene, 21:77-85 (1983) for further
details.
For the present invention, the imrnobile phase
of the hybridization system can be a series or matrix of
spots of known kinds and/or dilutions of denatured DNA.
This is most simply prepared by pipetting appropriate
small volumes of native DNA onto a dry nitrocellulose or
nylon sheet, floating the sheet on a sodium hydroxide
solution to denature the DN~, rinsing the sheet in a
neutralizing solution, then baki.ng the sheet to fix the
DNA. ~efore DN~:DNA hybridization, the sheet is usually
treated with a solution that inhibits non-specific
binding of added DNA during hybriclization.
T}lis invention involves the labelin~ of whole
genomic DNA, whole nucleic acids present in cells, whole
cell lysate, or unlysecl whole cells. Once the labeled
material is prepared, it can be used for the detection,
i.e., the presence or absence of certain specific genomic
sequences by speclfic nucleic acid hybridization assays.
One method according to the invention involves
the separation of celis from a human sample or the human
sample directly i.s treated by mixing with a
photochemically reactive nucleic acid binding
intercalating ligand. The mixture is incubated depending
on the type of th~ sample. If the sample is lysed cells
or nucleic acids, it is incubated for a period between a
few seconds to five minutes and when whole cells or
par~ial].y lysed cells are used, incubation between two
~3~ ~7~
minutes to two hours is employed. After the mixing and
incubation, the whole sample mixture is irradiated at a
particular wavelength for the covalent interaction
between the photochemically reactive DNA binding ligand
and the test sample. Then this labeled material is
hybridized under speciEic hybridization condi.tions with a
specific probe.
After the hybridization, the unreacted
unhybridized labeled test sample is removed by washing.
.~fter the washing, the hybrid is detected through the
label carried by the test sample, which is specifically
hybridized with a specific probe.
The present invention is surprising since in a
human genomic sample the amount of a single copy gene is
very low, for example, if a restriction fragment of one
thousand base pairs is the region of hybridization, the
frequency of such sequence in the whole human genomic
sample is one in a million. This conclusion has been
derived by assuming from ~he literature that a human
genomic sample has 3 x 109 base pairs and 1000 base pairs
will be 1/3,000,000 of that number. Automatically, in a
sample of human D~A containing approximately five to ten
micrograms of nucleic acids, only 5 to 10 picogram of the
corresponding sequences is available and labeling the
vast majority of the non-specific DNA should produce more
background than the true signal. But after the reaction,
i~ is surprising to observe that the results are not only
specific, but also of unexpected higher sensitivity.
Without wishing to be bound by any particular
theory of operability, the reason for the unexpected
sensitivity may be due to the formation of a network of
non-specific nucleic acid hybrids bound to the specific
hybrid, thus amplifying the amount of the signal. As has
been shown in a typical e~ample, a 19 nucleotide long
spcciEic seyuence containing plasmld i5 imrnobilized and
~31~79~
hybridized with 5 microgram equivalent of a test sample
which is labeled photochemi.cally and one detects very
easily the signal resulted from such hybrid. This could
not have been accomplished by any other technique because
of the problems associated with tile labeling method.
The present invention relates to a novel
hybridization technique where probes are immobilized and
an eukaryotic nucleic acid sample is labeled and
hybridized with immobilized unlabeled probe. A
surprising characteristic of the invention is the ability
to detect simple or multiple copy gene defects by
labeling the test sample. Since there is no requirement
for an excess of labeled hybridizing sequence, the
present method is more specific. In the present
invention, simultaneous detection of different gene
defects can be easily carried out by immobilizing
specific probes.
For example, using the present invention, one
can immobilize oligonucleotide probes specific for
genetic defects related to hemoglobinopathines, such as
sickle cell anemia and alp}-a-thalassemias on a sheet of
nitrocellulose paper, label the test sample and hybridize
the labeled test sample with the immobilized probes. It
is surprising tl~at partially purified or unpurified
nucleic acid samples (cell lysate or whole cell) can be
photochemically labeled with sensitive molecules without
af~ecting the specific hybridizability.
The oligonucleotide can be cloned in cloning
vectors, e.g., ~1 13, PUC 19 and PBR and accordingly the
vector containing the oligonucleotide acts as the probe.
The present invention is also directed to
detecting eukaroytes (protists) in samples from higher
organisms, such as animals or humans.
l~ukaroyte.c, include alyae, protozoa, fungi and
slime molds.
24
13~7~
The term "algae" refers in general to
chlo~ophyll-containing protists, descriptions of which
are found in G.M. Smith, Cryptogamic Botany, 2nd ed. Vol.
1, Algae and Fungi, McGraw-Hill, (1955).
Eukaryotic sequences according to the present
invention includes all disease sequences except for
bacteria and viruses. Accordingly, genetic diseases, for
example, would also be embraced by the present invention.
Non-limiting e~amples of such genetic diseases are as
~ollows:
Area Affected Diseases
Metabolism Acute intermitten-t porphyria
Variegate porphyria
alpha1-antitrypsin deficiency
Cystic fibrosis
Phenylketonuria
Tay-Sachs disease
Mucopolysaccllaridosis I
Mucopolysaccharidosis II
Galactosaemia
Homocystinuria
Cystinuria
Metachromic leucodystrophy
Nervous System Huntington's chorea
~eurofibromatosis
Myotonic dystrophy
l'uberous sclerosis
Neurogenic muscular atrophies
Blood Sickle-cel]. anaemia
Beta thalassaemia
Congenital spherocytosis
llaernopllilia A
Bo~el Polyposis coli
Kidney Polycystic disease
Eyes Dominant blindness
Retinoblastoma
Ears Dominant early childhood deafness
Dominant otosclerosls
Circulation Monogenic hypercholesterolaemia
slood Congenital spherocytosis
Teeth Dentinogenisis imperfecta
Amelogenisis imperfecta
Skeleton Diaphysial aclasia
Thanatophoric dwarfism
Osteogenes imperfecta
Marfan syndrome
Achondroplasia
Ehlers-Danlos syndrome
Osteopetrosis tarda
Cleft lip/palate
Skin Ichthyosis
Locomotor Muscular dystrophy
A nucleic aci.d probe in accordance ~ith the
present invention is a sequence which can determine the
sequence of a test sample. The probes are usually DNA,
RNA, mixed copolymers of ribo- and deoxyribonucleic
~ci.d5, oligonucl(~o~ides cor)~aining riborlucleotides or
26
131~7~4
deo~yribonucleotide residues or their modified forms.
The sequence of such a probe should be complementary to
the test sequence. The extent of complementary
properties will determine the stability of the double
helix formed after hybridiæation. The probe can also
have covalently linked non-complementary nucleic acids.
They can serve as the sites of the labeling reaction.
The nucleic acid is preferably labeled by means
of photochemistry, employing a photoreactive DNA-binding
furocoumarin or a phenanthridine compound to link the
nucleic acid to a label which can be "read" or assayed in
conventional manner, including fluorescence detection.
One use of the present invention is the
identification of bacterial species in biological fluids.
In one application, samples of urine from subjects having
or suspected of having urinary tract infections can
provide material for the preparation of labeled DNA(s) or
RNAs, while a solid support strip, e.g., made of
nitrocellulose or nylon, can contain individual dots or
spots of known amounts of denatured purified DNA from
each of the several bacteria likely to be responsible for
infection.
The format of labeled unknown and unlabeled
probes, which is the converse of standard schemes, allows
one to identify among a number of possibilities the
species of organism in a sample with only a single
labeling. It also allows simultaneous determination of
the presence of more than one distinguishable bacterial
species in a sample (assuming no DNA in a mixture is
discriminated against in the labeling procedure).
I{owever, it does not allow in a simple way, better than
an estimate of the amount of DNA (and, therefore, the
concentration of bacteria) in a mixed sample. For such
quantitation, sample DNA is immobilized in a series of
27
131~7~
dilution spots along with spots of standard DNA, and
probe DN~s are labeled.
A urinary tract infection is almost always due
to monoclonal growth of one of the following half dozen
kinds of microorganism: Escherichia coli (60-90~ of
UTI), Proteus spp. (5-20% of UTI), Klebsiella spp (3-10
of UTI), Staphylococcus spp. (4-20~ of ~TI),
Streptococcus spp. (2-5% of UTI). Pseudomonas and some
other gram negative rods together account for a low
percentage of UTI. ~ common contaminant of urine samples
that is a marker o~ improper sample collection is
Lactobacillus.
The concelltration of bacteria in a urine sample
that defines an infection is about 105 per milliliter.
The format for an unlabeleci probe hybridization
system applicable to urinar~ tract infections is to have
a matrix of DN~s from the above list of species,
denatured and immobilized on a support such as
nitrocellulose, and in a range of amounts appropriate for
concentra~ions of bacterial DNAs that can be expected in
samples of labelled unknown.
Standard hybridization with biotinylated whole
genome DNA probes takes place in 5-10 ml, at a probe
concentration of about 0.1 /ug/ml; hybridization oE probe
to a spot containing about 10 ng denatured DNA is readily
detectable. There is about 5 fg of DNA per bacterial
cell, so that for a sample to contain 1 Jug of labeled
DNA, it is necessary to collect 2 x 108 bacteria. If an
infection produces urine having approximately 105
bacteria/ml, then bacterial DNA to be labeled from a
sample is concentrated froM 2000 ml. If more than 10 ng
unlabeled probe DNA is immobilized in a dot, for example,
100 nq or 1 /ug, or if the hybridization volume is
reduced, then the volume of urine required for the
28
- ~3~79~
preparation of labeled unknown is approximately a few
tenths of a ml.
A strip o~ dots containing immobili~ed,
denatured, unlabelled probe DNAs could have the following
conLiguration:
1 ~Ig 10 ng 100 pg
Escherichia o o o
Proteus
Klebsiella o o o
Staphylococcus o o o
Streptococcus o o o
Pseudomonas o o o
Lactobacillus o o o
This procedure involves the labeling of DNA or
RNA in a crude cell lysate. Ideally, preparation of
labeled sample DNA or RNA will accommodate the following
points:
(1) bacteria will be concentrated from a fluid
sample by centrifu~ation or filtra-tion;
(2) bacteria will be lysed under conditions
sufficient to release nucleic acids from the most
refractory of the organisms of interest;
(3) the labeling protocol will not re~uire
purification of labeled nucleic acids from unincorporated
precursors, nor the puri'ication of nucleic acids prior
to labeling;
(4) the labelinq protocol will be sufficiently
specific for DNA and/or RNA that proteins, lipids and
polysaccharides in the preparation ~Jill not interfere
with hybridization nor read-out.
In the present invention, there is provided a
method for efficiently and rapidly lysing whole cells,
including Gram posi~ive bac~eria. The rnethod involves
29
~L31~7~
contacting cells, e.g., whole cells, with an alkali,
e.g., sodium or potassium hydroxide solu~ion in a
concentration of 0.1 to 1.6 Normal.
The important fea-tures of the present lysis
protocol are its relative simplicity and speed. It
employs a common chemical that requires no special
storage conditions and it lyses even Gram positive
organisms with high efficiency, while preserving the
proper~ies of the DNA that are important for subsequent
steps in the photochemical labeling process.
For the present invention, the i.mmobile phase
of the hybridization system can he a series or matrix of
spots of known kinds and/or dilutions of denatured DNA.
This is most simply prepared by pipet-ting appropriate
small volumes of native DNA or oligonucleotides onto a
dry nitrocellulose or nylon sheet, floating the sheet on
a sodium hydroxide solution to denature the DNA, rinsing
the sheet in a neutralizing solution, then baking the
sheet to fix the DNA. Before DNA:DNA hybridization, the
sheet is usually treated with a solution that inhibits
non-speci~ic binding of added DNA during hybridization.
The invention will be further described in the
following non-limiting examples wherein parts are by
weight unless otherwise expressed.
Example 1: Preparation of ~abeling Compound
The preparation of the labeling compound
required 1-amino-17-N-(Biotinylamido)-3,6,9,12,15
pentaoxaheptadecane. This compound was prepared in the
following four steps:
(a) 3,6,9,12,15 pentaoxaheptadecane
1,17-diol ditosylate was synthesized.
(~) 1,17-~.ipthalimido deriva~ive of 3,6,9,12,
l5 pentaoxaheptadecane was prepared.
~ 3~7~
(c) 1,17-diamino derivative of 3,6,9,12,15
pentaoxaheptadecane was prepared.
(d) 1-amino, 17-biotinylamido derivative of
3,6,g,12,15 pentaoxahep~adecane was
prepared.
xample l(a): Preparation of 3,6,9,12,15-Pentaoxahepta-
decane-1,17-diol Ditosylate
To a stirred solution containing 50 g of
hexaetllylene glycol (0.177 mol) and 64 ml of
triethylamine (39.36 g, 0.389 mol) in 400 ml of CH2C12 at
0C was added dropwise a solution containing 73.91 g of
p-toluenesulfonyl chloride (0.389 mol) in 400 ml of
CH2C12 over a 2.5 hour period. The reaction mixture was
then stirred for one hour at 0C and then heated to
ambient temperature for 44 hours. The mixture was then
filtered and the filtrate was concentrated in vacuo. The
resulting heterogeneous residue was suspended in 500 ml
o~ ethyl acetate and filtered. The filtrate was then
concentrated in vacuo to a yellow oil which was
triturated eight times with 250 ml portions of warm
hexane to remove unreacted p-toluenesulfonyl chloride.
The resulting oil was then concentrated under high vacuum
to yield 108.12 g of a yellow oil (quantltative yield).
nalysis: Calculated for C26H38O11S2
Calc.: C, 52.87; H, 6.48.
found: C, 52.56; H, 6.39.
PMR: (60 MHz, CDC13) ~ : 2.45 (s, 6H); 3.4-3.8 (m~ 20H);
~.2 (m, 4H); 7.8 (AB quartet, J=8Hz, 8H).
IR: (neat) cm 1 2870, 1610, 1360, 1185, 1105, 1020,
930, 830, 785, 670.
31
13~7~
Example 1 (b): Preparation of 1,17 Diphthalimido-
3,6,9,12,15-pentaoxaheptadecarle
A stirred suspension eontaining 108 g of
3,6,9,12,15-pentaoxaheptadecane- 1,17-cliol ditosylate
(0.183 mol), 74-57 g of potassium phthalimide (0.403
mol), and 700 ml of dimethylaeetarnide was heated at
160-170 C for 2 hours and was then coo].ed to room
temperature. The precipitate was filtered and washed
with water and acetone to yield 53.05 g of product as a
white powder which was dried at 55C (0.1 mm) . mp
124-126C.
A second crop of product was obtained from the
dimethylacetamid~ filtrate by cvaporatioJI in vacuo and
the resultiny precipitate with was successively washed
ethyl acetate, water, and acetolle. The resulting white
powder was dried at 55C (0.1 mm) to yield an additional
9.7 g of product. mp 124.5-126.5C. The combined yield
of product was 62:82 g (68~ yield) .
nalysis: (For first crop)
Calculated for C28~332N29 1/2~320
Calc.: C, 61.19; H, 6.05; N, 5.09.
found: C, 61.08~ ll. 6.15; N, 5.05.
(For second erop)
Calculated for C28H32N2O9
Calc.: C, 62.21; H, 5.97; N, 5.18.
found: C, 61.78; 13, 6.15; N, 5.13.
PMR: (60 MHz, dmso-d6) ~: 3.5 (s, 81-1); 3.6 (s, 8H); 3.8
(bt, J=3Hz, 8H): 8.1 (s, 811) .
IR: (KBr) cm : 2890, 1785, 1730, 1400, 1100, 735.
~L31 ~79~
xample l(c): Preparation of 1,17-Diamino-3,6,9,12,15-
Pentaoxaheptadecane
A solution containing 60 g of
1,17-diphthalimido-3,6,9~12,15-pentaoxaheptadecarle (0.118
mol), 14.8 g of hydra~i.ne hydrate (0.29G mol), and 500 ml
of ethanol were hc-ated with mechanical stirring in a
100C oil bath for three hours. The mixture was then
cooled and filtered. The resultant filter cake was
washed four times with 300 ml portions of ethanol. The
combined filtrates were concentrated to yield 32.35 g of
a yellow apaque qlassy oil. The evaporative distillation
at 150-200C (0.01 mm) gave 22.82 g of a light yellow oil
(69% yield). lit. b.p. 175-177C (0.07 mm).
PMR: (60 MHz, CDC13) ~ : 1.77 (s, 4H, NH2);
2.85 (t, J=5Hz, 4H); 3.53 (t, J=5Hz, 4H); 3.67
(m, 16H).
IR: (CHC13) cm : 3640, 3360, 2860, 1640, 1585, 1460,
1350, 1250, 1100, 945, 920, 870.
Mass Spectrum: (EI) m/e = 281.2 (0.1%, M+1).
(FAB) m/e = 281.2 (100%, M+1).
12 28 2 5-1/2 H~O
Calc.: C, 49.80, I1, 10.10; N, 9.68.
~ound: C, 50.36; II, 9.58; N, 9.38.
Literature Re~erence: W. Kern, S. Iwabachi, H. Sato and
V. Bohmer, Makrol _Chem., 180, 2539 (1979).
ple l(d): Preparation of_1-Amino-17-N-(Bi.otinyl-
amido?-3 ! 6,9,12,15-pent:aoxaileptadecane
. _
A solution contalning 7.2 y of
1~l7-dia~.no-3~9~l2~]s-p~ntaoxaheptadecane (25 rnmol) in
75 ml of DMF under an aryon atmosphere was treated with
33
13~ ~7~
3.41 g of N-succinimidyl biotin (10 mmol) added in
portions over 1.0 hour. The resulting solution was
stirred for four hours at ambient temperature. TLC
(SiO2, 70:10.1 CHCL3-CH3OH-cone. NH4 OH) visualized by
dimethylaminocinnamaldehyde spray reayent showed
exeellent conversion to a new product (Rf=0.18). The
reaction mixture w~s divided in half and each h~lf was
absorbed onto SiO2 and flash-ehromatographed on 500 g of
SiO2-60 (230-400 mesh) usiny a 70:10.1 CHCl3-C~3O~I-eonc.
NH40H solvent mixture. Fractions eon~aining the product
were polled and concentrated to a yield 2.42 g of a
gelatinous, waxy solid. The product was precipitated as
a solid from isopropanol-ether, washed with hexane, and
dried at 55C (0.1 mm) to give 1.761 g of a white powder
(35~ yield).
nalysis: Calculated for C22H42N4O7S.3/2 H2C:
C, 49.51; H, 8.50; N. 10.49.
found: C, 49.59; H, 8.13; N, 10.39.
MR: (90 MHz, dmso-d6)~ : 1.1-1.7 (m, 6H)i 2.05 (t,
J=711z, 2H);
2.62 (t, J=4Hz, lH); 2.74 (t, J=4Hz, lH)i 3.0-3.4
(m, 14H).
3.50 (s, 141-1); 4.14 (m, lH); 4.30 (m, lH); 6.35 (d,
J=4Hz, lH); 7.80 (m, lH).
CMR: (22.5 MHz, dmso-d6)~ : 25.2, 28.0, 28.2, 35.1,
40.6, 55.3, 59.2, 61.1, 69.6, 69.8, 71,2, 162.7,
172.1.
IR: (KBr) cm : 2900, 2350, 1690, 1640, 1580, 1540,
1450, 1100.
Mass ~,pectrurn (FA13) m/e: 507 . 3 (M~l, 56% )
34
... . .. .. _ . ., .... , .. . __ .. ... . _ .. _._.. ... ..... _ .-- .. . .... _ _ .. _ .. _ __ _ .__., ._.__ _ . _ _
~3~47'~
Example 2: Preparation of 4'-Biotinyl-PEG-4,5'-
dimethylangelicin
-
A solution of 203 mg of 1-amino-17-N-~biotinyl-
amido)-3,6,9.12,15-pentaoxaheptadecane (0.4 mmol) in 1 ml
of DM~ under an argon atmosphere was trea~ed with 78 mg
of N,~-carbonyldimidazole ~0.48 mmol). Tl-e resulting
mi~ture was stirred for four hours and was then treated
with 55 mg of 4'-aminomethyl-4,5'dimethylingelicin
hydrochloride (0.2 mmol), 140 ~ll of diisopropylethyl-
amine, and 100 ~1 of DMF`. The resulting mixture ~as
stirred overnight at 50C. The mixture was then
evaporated onto SiO2 in vacuo and the resultant
impregnated solid flash was chromatographed on 60 g.of
SiO2 (230-900 mesh) eluted with 1.5 liters of 7%
CH3-CHC13 followed by 1 liter of 10~ CH3OH-CHC13.
Fractions containing the product were pooled and
concentrated to yield 72 mg of a glassy solid (47
yield).
PMR: (90 MH~, dmso-d6):~ 1.1-1.8 (m~ 6H); 2.04 (bt,
J=7Hz, 2H); 2.5 (s, 6H); 2.56 (m, lH); 2.74 (bd,
J=4Hz, lH); 2.8-3.4 (m, 14H); 3.40 (m~ 14H); 4 14
tm, lH); 4.25 (m, lH); 4.40 (bd, J=6Hz, 211); 6.5
(m, lH); 6.35 (s, lH); 7.02 (s, lH); 7.45 (d,
J=8Hz, lH); 7.62 (d, J=8Hz, lH); 7.80 (m, lH).
C~lR: (22.5 MHz, dmso-d6) ~ : 11.9, 18.9, 25.3, 28.2
28.3, 33.4, 35.2, 55.4, 59.2, 61.0, 69.2, 69.~,
69.8, 70.0, 89.0, 107.8, 112.0, 113.1, 114.3,
120.6, 121.6, 153.6, 154.4, 155.6. 157.9, 159.5,
162.7, 172.1.
Literature Refer~nce: F. Dall'Acqua, D. Vedaldi, S.
Caffieri, ~. Guiotto, P. Rodighiero, F. Baccichetti, F.
Carlassare and F. Bordin, J. Med Chem., 24, 178 (1981).
.. ....... ~.. __ .... .. . ..... ~ .__ .____ _
131479~
Example 3: Colorimetric or Chemiluminescent Detection of
the Nuclei.c Acid Hybrids
E~ample 3(a): Colorimetrlc Detection
____
Colorimetric detection of the biotinylated
hybrids is carried out following the procedure and kit
developed ~y Bethesda ~esearch Laboratories (B~L),
Gaithersburg, Maryland 20877, U.S.A. The procedure is
descrihed in detail in a manual supplied with a kit by
B~L, entitled "Products for Nucleic Acid Detection", "DNA
Detection System Instructlon Manual", Catalogue No.
8239SA.
.
Example 3(b): Chemiluminescent Detection
. . .
Chemiluminescent detection of the biotinylated
hybrids is identical to the above method: the filters
with the hybrids are saturated with BSA (bovine serum
albumin) by immersing the paper in 3% BSA at 42C for 20
minutes. Excess BSA is removed by taking the paper out
of the container, and blotting it between two pieces of
filter paper. The paper is then incubated ln a solution
containing Streptavidin (0.25 mg/ml, 3.0 ml total
volume), for 20 minutes at room temperature. It i5 then
washed three times with a buffer containing 0.1 M
Tris-HCl, p~l 7.5, 0.1 M NaCl, 2 mM MgCl2, 0.05% "TRITON
X-100". Next the filter is incubated with biotinylated
horseradish peroxidase (0.10 mg/ml) for 15 minutes at
room temperature. This is ~ollowed by three washings
with 0.1 M Tris-HCl, pH 7.5, 0.1 M NaCl, 2 mM MgCl2 and
0~05% Triton X-100, and one washing with 10 mM Tris (pH
8.0) buffer. Chemiluminescent activation is conducted in
two ways. (1) Spots are punched out and the discs
containing the DNA are placed in a microtiter plate with
wells that are painted black on the sides. After the
punched paper circ].e.~ arc placed in the microtiter plate
36
`b~
~3:L~7~
wells, 0.8 ml buffer containing 40 mM Tris and 40 mM
ammonium acetate (pH 8.1) is added to each well. Then 10
~1 of 1:1 mixture of 39 mM Luminol (in DM~) and 30 mM
~2~ (in wa~er) is added. Light emission is recorded on
a "POLAROID" instant film by exposing it direc~ly in the
film holder. Alterna~ively (2), the paper is soaked in a
solution containing 1:1 mixture of 0.5 mM Luminol and
H202 and wrapped with a transparent "SARAN WRAP". The
light e~ission is recoxded on a "POLAROID" film as above.
Example 4: General Method of Labeling the Test Sa~ple
Nucleic Acids
.. .. . .
Hi~h molecular weight DNA from a patient's
sample is isolatecl by a method described in U.S.P.
4,395,486 (Wilson et al),
_ The nucleic acid
is dissolved in 10 mM borate buffer (p~l 8.0) to a final
concentration of approximately 20 ~g/ml. To the nucleic
acid solution "angelicin-peg-biotin" in aqueous solution
is added to a final concentration of 10 ~g/ml. The
mixture is then irradiated at long wavelength irradiation
for about 60 minutes using a black ray UVL 56 lamp. The
product is ready for hybridization without purification.
~owever, the product can be purified by dialysis or
alcohol precipitation (~.S.P. 4,395,486) as is usually
followed for nucleic acids.
Instead of nucleic acids, whole cell lysate can
also be labeled following an identical procedure. The
lysis is conducted by boiling the cells with 0.1 N sodium
hydro~ide, followed by neutralization with hydrochloric
acid.
When whole cells are used, the mixture of
"PEG-ang-bio" and cells are incubated for at least 60
minutes prior to irradiation for effi.cient tran~port of
*Trade Mark
A
7 ~ ~
the ligands Many different variations of the above
described methods can be adopted for labeling.
E~ample 5:
Alpha-thalassemia is associated with gene
deletion. The detection of gene deletion by hybridization
in a dot/slot blot format requires that the total amount
of sample and its hybridizability are accurately known.
Since the beta-globin gene is a slngle copy gene,
simultaneous hybridization of a sample with beta-globin
and alpha-globin and their relative amounts will indicate
the amount of alpha-globin with the sample.
The format and hybridization conditions are the
same as Rubin and Kan, supra, except probes, not test
VNA, is immobilized. }Iybridization conditions are also
similar. The detection is done by using the BRL kit
described supra following BRL's specifications.
The hybridization detection process are
conducted in three steps as follows:
Step 1: Immobilization of the Probes
As described in Rubin and Kan, supra, 1.5 kb
PstI fragment containing alpha2 globin gene is used as a
probe for alpha-thalassemia and for the beta-globin gene
a 737 base pair probe produced by the digestion of pBR
beta Pst (4.4 kb) is used. The beta-globin gene probe
has been described in U.S.P. 4,395,486 (column 4). For
the detection of gene deletion related -to
alpha-thalassemia, the amount of starting nucleic acid,
hybridization efficiency and control samples are needed.
The present invention avoids these problems by
simultaneous hybridization with a single copy essential
gene (e.g., beta-globin gene) when simi.lar amounts of
probes are immobilized side by sid~, labeled sample is
hybri.dized, ~ tive strength of signal intensity is a
~3~ ~7~
measure of relative amount of gene dosage present in the
sample.
The probes (0.5, 1, 3 and 5 ~ per 100 ~1) are
suspended in 10 m~ tris HC1 ~pH 7) buffer, denatured with
20 ~1 3 M sodium hydroxide, at 100C, for 5 minutes, an
equivalent volume of 2 M ammonium acetate, pH 5.O is
added to neu~ralize the solution, immediately after
neutralization the probes for beta- and alpha-globin
genes are applied in parallel rows to nitroc~llulose
filter paper under vacuum in a slot blot manifold,
purchased from Scleicher and Schuell, ~Keene, New
Hampshire, U.S.A.). The filter is then dried in vacuum
at 80C for 60 minutes. It is then prehybridiz~d for 4
hours in a mixture containing 50 mM sodium phosphate (pH
7) 45 mM sodium citrate, 450 mM sodium chloride, 50%
~v/v) formamide, 0.2% each ~w/v) of polyvinyl
pyrrolidine, "FICOLL 400" and bovine serum albumin and
0.2 mg/ml alkali boiled salmon sperm DNA and 0.15 mg/ml
yeast RNA.
Step 2: Lab_ling of the Test Sample
This was described above.
Step 3: Hybridization
The nitrocellulose strip containing the
immobilized probes are hybridized with the labeled test
sample in plastic ba~s (e.g., "SEAL-A-MEAL~', "SEAL and
SAVE'', etc.J. ~Iybridization solution is the same as
prehybridization solution plus 10% dextran sulphate.
Hybridization is done at 42C for 16 hours. After
hybridization detection of biotin is conducted with a kit
and procedure supplied by ~ethesda Research Laboratory,
Maryland, U.S.A., (catalogue No. 8239SA). Results of
relative intensity of alpha- and beta- regions are used
to estimate the exterlt o~ dele~ion o~ alpha-globin genes:
*Trade Mark
.. ..... .... .. ........ , ..... , ~ ....... . ... ... . ... . .. .
l3~-~r~
No signal on the alpha-globin side: all 4
alpha-globin genes missinq.
Signal on the alpha-globin side is half as
stronq as on the corresponding beta-side: 3 alpha-globin
genes missing.
Signals on alpha and beta side equivalent: 2
alpha-globin genes missing.
Signals on alpha side is s~ronger than the
corresponding ~eta side (2 alpha = 3 beta):
alpha-globin gene missing.
Example 6: Immobilization of an Oli~onucleotide Sequence
Specific for Hemoq]obin Mutation
It is known that an oligonucleotide cannot be
easily immobilized onto nitrocellulose paper by a simple
adsorption process. The present invention encompasses
three different methods to incorporate an oligonucleotide
sequence into a larger molecule capable of adsorption.
~ethod 1: Two oligonucleotides, one a 43mer and the
other a 16-mer, have been chemically synthesized in an
automated synthesizer (Applied Biosystem 3gOB) by the
phosphoramidite-method and phosphorylated at the 5' end
by a T4-polynucleotide kinase mediated process according
to Maniatis et al, Molecular Clonlng, page 122. These
oligonucleotides contain a segment of a 19 nucleotide
long sequence specific ~or the detection of the mutation
associated with sickle cell anemia.
43mer A & S (A = normal globin gene; S = sickle
globin gene) were kinased according to Maniatis et al,
Molecular Clonlng, page 122, in two separate reactions,
namely, one with 3 P-ATP and one with no radioactive
label. 0.4 ~g 32P-43mer and 0.6 mg cold 43mer were mixed
and purified on a spun column (G-25med in TE (Tris EDI`A
buf~er~) to ~ ~inal volume of ~O Jul. I'wo dilutiolls were
~3~7~
spotted on S & S (Schleicher & Schuell) nitrocellulose
and nytran (nylon) membranes at 50 and 0.5 ng.
Method 2: The phosphorylated oligonucleotide products of
method 1 were further elongated by making multimers of
sequences by a ligase mediated process. The principle is
described as follows:
(X)
. _ . . . _
3' 5' 3' 5'
ligase
\l /
. .
3' 5'
strand separation
\ ,
. _ _
3' 5'
.
(X)
The product being o~ a higher molecular weight
than an oligonucleotide it should be immobiliza~le by
adsorpt:ion on to a nitrocellulose paper.
41
~3~79~
Aqueous soluti.ons containing 4~g of 32P43mer
and 3.7 lug 16mer linker (X) were mixed and dried under
vacuum. 6 mg of cold kinased 43mer was added and the
sample was heated to 55C and cGoled slowly to 0C to
anneal. Liqation was carried ou-t in 20 ~l total reaction
volume with 800 units of ligase (Pharmacia) at 15C for 4
hours. 1 mg (2 ~l) was purified on a spun column
(G-25med in T~) to a final volume of 40 ~l. Two
dilutions were spotted on nitrocellulose nylon membranes
at 50 and 0.5 ng.
~1ethod 3: The same as method 2, but ligation was not
conducted. Instead of liyation, cross linking was
conducted with an intercalator to keep the double
stranded regions intact. Hence, the cross linked
molecule will have several oligonucleotide sequences
covalently linked to each other.
2 lug of 32P43mer (for sequence P-50) was added
to 2.9 mg of a l6mer (for sequence P-50) linker and
purified on a spun column (G25med in TE) to a final
volume of 40 ~l. 6 mg of kinased 43mer was added and the
samples were heated to 55C and cooled slowly to 0C to
anneal. 25 ~1l of intercalation compound
aminomethyltrioxsalen was added and the sample was
irradiated for 30 minutes on ice in 500 ~l total 10 mM
borate buffer pH 8.2 with a long wave UV lamp model
(UVL-21, ~ = 366 nM).
The probes modified by all three methods were
then immobilized on to nitrocellulose and nylon paper and
hybridized with labelled oliyonucleotides. The results
indicate that the sequence are immobilizable and
hybridization fidelity remains intact.
Two dilutions of the products of rnethods 1 to 3
were spotted on nitrocellulose and nylon membranes at 50
and 0.5 ngs.
42
. . ~, . .:.;
.,.. ; - ~
..... :. `:.. .
1 3:~7~
Whole filters were baked for 30 minutes in 80C
vacuum oven and prehybr-dized in blotto ~5~ nonfat dry
milk, 6XSSC, 20 mM Na-pyrophospllate) for 30 minutes in
50C oven.
Hybridization was carried out with primer
extended l9'A ~ l9S' probes at 50C for one hour (3
strips/probe).
Filters were s-tringently washed for 15 minutes
at room temperature in 6XSSC with slight agitation and 2
x 10 minutes at 57C.
Air-dried filters were place on Whatman paper
and autoradlographed at -70C overnight.
The results presented in Fig. 1 surprisingly
indicate specific hybridization are obtained by
immobilizing oligonucleotide probes.
Example 7: H~/bri~ization with labeled gerlomic DNA for
~on ~adioactive Detection
.
Human normal genomic (~X) DNA was photolabeled
with "biotin-PEG-angelicin" (BPA) in 10 mM borate buffer
pH 8.2 at a weight ratio of 0.3 to 1 (~PA:DNA) for 15
minutes on ice with a long wave UV lamp model UVL-21,
J~= 366 nm. No puri~ication is necessary.
Target DNA oligonucleotides were directly
immobilized on S & S nitrocellulose in 1 ~1 aliquots at
the following concentratioll, and then baked in an \30C
vacuum oven for 30 minutes. The amounts of the different
in~obilized probes are as follows:
.3-mer (A) - Kinased (method 1) 200 ng
43-mer (A) 200 ng
43-mer (S) - Kinased (method 1) 200 ng
43-mer (S) 200 ng
~11319Ass 50 ng
M1319Sss 50 ng
M]3737~ss 50 ng
43
1 3~ 7~4
sRL Co~nercially biotinylated DNA 200 pg
pUC19 50 ng
43-merA: 5' GGAT3AAT4CTCCTGAGGAGAAGTCT
GCT4AATCTTAA 3'
* =T for 43-mer S
16-mer (Common to both A and S)
3' - TTAGAATTCCTAAATT-5'
Filters were prehybridized in blotto (5% nonfat
dry milk, 6XSSC, 20 mM Na-pyrophosphate) for 30 minutes
in a 45C H2O bath.
All 4 strips were ilybridized in 2 mls solution
containing 2 /ug laheled XX DNA containing normal
beta-ylobin gene (hybridization solu~ion was blotto with
10~ PEG) for 2 hours in 45C in a 112O bath.
A stringency wash was carried out as follows:
1 X 20' at room temperature in 6XSSC
2 X 20' at temperatures indicated in Fig. 2
witii very little agitation.
50 ml centrifuge tubes were used for elevated
temperature washes. ~ecults are shown in Fig. 2.
Detection of biotin in the hybrid was carried
out according to the Bethesda ~esearch Laboratory,
Bethesda, Maryland, ~.S.h., manual using their kit for
biotin detection. The results indicated specific
hybridization.
E~ample_8: Immobilization of Whole Genomic_DNA As Probes
Tens of milligram to gram amounts of DNA were
prepared in the following manner ~rom bacterial cells
harvested from fermentor cultures. Bac-teria were
collected by centrifugation from 10 liter nutrient broth
cultures grown in a Ncw Brunswic~ Scientific Microferm
44
Fermentor. Generally, cells in concen~rated suspension
were lysed by exposure to an ionic dete~gent such as SDS
(~a dodecyl sulEate), then nucleic aci~s were puri~ie~
from proteins and lipids by e~traction with phenol and/or
chloroform (J. Marmur, J. Mol. siol., 3, 208-218, 1961).
R~A was removed from the nucl~ic acids preparation by
treatment of the DNA solu~ion with 0.2 mg/ml ribonuclease
at 37C, then DNA was precipitated from solution by the
addition of two volumes of ethanol. Bacterial DNA
redissolved from the precipitate in a low salt buffer
such as TE (10 mM Tris-HCl, pH 7.5, 1 n~ Na2 EDTA) was
characterized with respect to purity concentration and
molecular size, then approximately 1 microgram aliquots
were denatured and immobilized as spots on nitrocellulose
or nylon membranes for hybridization (Kafatos et al.,
Nuclelc_Acids, Res. 7, 15~1-1552, (1979)). Denaturation
was accomplished by exposure of the DNA with
approximately 0.1 N NaOH. After ~enaturation the
solution was neutralized, then the Inembrane was rinsed in
NaCl/Tris-HCl, p~l 7.5, and dried.
Example 9: Processing of a Test Sample for Cellular DNA
Labeling
Samples of urine, for example (although the
following can equally apply to suspensions of material
form gonorrhea-suspect swabs, from meningitis-suspect
cerebrospinal fluid, frorn contamination-suspect water
samples, etc.), are centrifuged or filtered to wash and
concentrate any bacteria in the sample. The bacteria are
then lysed by exposure to either (i) 2 mg~ml lysozyme or
lysostaphin then exposure to approximately 90C heat,
(ii) 0.1 to 1.6 N NaO~I, or (iii) 1~ Na dodecyl sulfate.
After (ii) NaOH, the cell lysate solution is neutralized
before labelling; after (iii) detergent lysis, DNA
l~be]lincJ is preceded by removal o~ the SDS with 0.5 M K
7~4
acetate on ice. Angelicin should be able to permeake
intact cells so that DNA labeling can be accomplished
before cell lysis. This in situ labeling simplifies the
e~traction procedure, as alkaline or detergent lysates
can be incorporated directly into a hybridi~ation
solution.
Prior to hybridi~ation, the labeled sample is
denatured, and it should also preferably be reduced to
short single stranded lengths to facilitate specific
annealing with the appropriate unlabeled probe DN~.
~.ethods of denaturation are ~nown in the art. These
methods include treatment with sodium hydroxide, organic
solvent, heating, acid treatment and combinations
thereof. Fragmentation can be accomplished in a
controlled way be heating the DNA to approximately 80C
in NaOH for a determined length of time, and this, of
course, also denatures the DNA.
Example 10: Labelin~ of the Products of Example 9
(i) A test sample of about 10ml urine will
contain 104 or more infectious agents. After separation
by centrifugation and washing, the pretreated cell lysate
(step 2) was resuspended in 0.2 ml 10 mM sodium borate
buffer (pH approximately 8). To this suspenslon, 10 ~g
of photolabelling reagent dissolved in ethanol (10
mg/ml), was added and mixed by shaking on a vortex mixer.
The rnixture was then irradiated at 365 nm for 30 minutes
with a UVGL 25 device at its long wavelength setting.
The UVGL device is sold by UVP Inc., 5100 Walnut Grove
Avenue, P.O. Box 1501, San Gabriel, CA 91778, U.S.A.
(ii) The sample was also labeled with
N-(4-azido-2-nitrophenyl)-N'-(N-d-biotinyl-3-
aminopropyl)-N'-methyl-1,3-propanediamine (commercially
available from BRESA, G.P.O. Box 498, Adelaide, South
46
131479~
~ustralia 5001, Australia), following the procedure
described by Forster et al (19~5), supra for DNA.
(iii) When unlysed cells were used, the cell
suspension in 0.~ ml 10 mM borate was incubatcd with the
photoreagent for 1 hour prior to irradiation.
.Yample 11: Hybridization of the Products of Examples 8
and 10
Prior to hybridi~ation, the membrane with spots
of denatured unlabeled probe DNA was treated for up to 2
hours with a "prehybridi~a~ion" solution to block sites
in the membrane itself that could bind the hybridization
probe. This and the hybridization solution, which also
contained denatured labelecl sample DNA, was comprised of
appro~imately 0.9 M Na , 0.1~ SDS, 0.1-5~ bovine serum
albumin or nonfat dry milk, and optionally formamide.
With 50~ formamide, the prehybridization and
hybri.dization steps were done at approximately 42C;
without, the temperature was approximately 68C.
Prehybridized membranes can be stored for some time. DNA
hybridization was allowed to occur ror about 10 minutes
or more, then unbound laheled DNA was washed from the
membrane under conditions such as 0.018 M Na (0.1 x
SSC), 0.1% SDS, 68C, that dissociate poorly base paired
hybrids. After posthybridization washes, the membrane
was rinsed in a low salt solution without detergent in
anticipation of hybridization detection procedures.
~ample_12: Detection of a Nucleic Acid Hybrid with
Inunu ogold
Affinity isolated goat antibiotin antibody
~purchased from Zymed Laboratories, San Francisco,
California, U.S.A.) was adsorbed onto colloidal ~old (~0
nm) following the method described by its supplier
(Jans:er, instruction booklet, Janssen Life Sciences
47
~ 3~7~
Products, Piscataway, New Jersey, U.S.A.) and reacted
with hybridized bio~inylated DNA after blocking as in a
colorimetric method. The signals were silver enhanced
using a Janssen (B2340 BEERSE, Belgium) silver
enhancement kit and protocol.
Example 13: Detection of Urinary Tract Infection ln a
Urine Sample
Urine samples were collected from a hospital
where they were analyzed by microbiological methods and
the results were kept secret until the hybridization
diagnosis was conducted. Then they were compared
ascertain the validity of the hybridization results.
1 ml of clinical sample (urine) suspected of
UTI infection was centrifuged in a Brinkman micro
centrifuge for 5 minutes. Then 0.1 ml of 1.2 N sodium
hydroxide was added and the suspension was heated to
100C to lyse the cells. The suspension was then diluted
to 1 ml with 10 mM sodium borate buffer p~ 8 and was
neutralized with hydrochlorine acid to a p~; of 7. To the
solution, 50 ~ug "biotin-PEG-anyelicin" (see Example 2) is
added and the mixture was irradiated with a UVL 56 long
wavelength UV lamp for 15 minutes. The irradiated sample
(0.1 ml) was added to 3 ml 3XSSC o~ 5~ nonfat dry milk
10~ PEG with 0.2 M sodium pyrophosphate and hybridization
was conducted with probes (whole genomic DNA) immobilized
onto nitrocellulose paper at 68C for 5 minutes to
overnight. After hybridization detection was conducted
according to Examples 3 or 12, the spots or the
photoyraphs were visually interpreted for the presence of
specific bacteria in the test sample. A spot of human
DNA was also present in the nitrocellulose paper for the
detection of leucocytes. The presence of leucocytes was
further verified wi-th a common method using "LEUKOSTIX"*
~Miles Laboratories, Elkhart, Indiana, U.S.A.).
~8
*Trade Mark
13~79~
Typical res~llts (Tables 1 and 2) indicate that
the hybridization diagnosis produces similar results in a
shorter time then the corresponding microbiological
assays. The present invention not only provides
information related to species identification, but also
the le~cocyte content in a clinical sample.
TABLE 1
DIAGNOSIS OF CLINICAL URINE S~PLES
_ _
* HOSPITALAPPLICANTS' HYBRIDIZATION DETECTION
DIAGNOSIS RESULTS SYSTEM
-
NEG NEG GOLD
NEG NEG GOLD
NEG NEG GOLD
NEG E.c.-M C~E~
NEG E.c.-VW GOLD
NEG E.c.-VW GOLD
____________________~___________________________________ __________
S+, C- N~ GOLD
S+, C- E.c.-S CHEMI
S+, C- E.c.-S, Kl.-M CHEMI
S+, C- NEG GOLD
S+, C- NEG GOLD
S+, C- NEG GOLD
S+, C- E.c.-VW GOLD
S+, C- NEG GOLD
S+, C- E.c.-VW GOLD
S+, C- NE~ GOI,D
S+, C- NEG - GOLD
_________ _________________________________________________________
100,000/mL E.c.E.c.-S GOLD
100,000/mL E.c.E.c.-S Cl~
100,000/mL E.c.E.c.-W GOLD
50,000/mL E.c.E.c.-M C~E~
50,000/mL E.c. NEG GOLD
E. coli E.c.-S, Kl.-M Cl~
E. coli E.c.-VS, Kl.-S CHEMI
E. coli E.c.-S, Kl.-S Cl~
E. coli/Klebsiella mix E.c.-S, Kl.-W GOLD
E. coli/Staph mix E.c.-S, St.-M CHEMI
__ _ _____________________________________________________________
~9
... . . _ ..... .... ..
79 ~
TABLE 1 (Continued)
DIAGNOSIS OF CLINIGU,URINE SAMPLES
*~IOSPITALAPPLIC~S'~DIZATION DETECTION
DIAGNCSIS RFAS TS SYSTEM
~lebsiella spp. E.c.-M, Kl.-W CH~
100,000/mL K. pneumoniae E.c.-W, Kl-VW GOLD
Fnterobacter spp. NEG** GOLD
100,000 Candida NE~** GOLD
100,000/mL Proteus Pr.-S, E.c.-W GOLD
_____________________________________________________ _____________
10,000/n~ Strep NEG ~E~I
Mixture of 3 unidentified ~n(-~) NEG GOLD
___________________________________________________________________
* diagnosis conducted ~y streaking urine on an agar plate
and treating the plate under condltions so that the
infectious organism can grow.
** Enterobacter/Candida probes not included in the
hybridization assay, t:herefore, negative results are not
surprising; given the high stringency conditiolls employed
in the assay, cross-hybridizati.on with species related to
Enterobacter was not detected.
Abbreviations: VS=very strong; S=strong; ~I=medium;
W=weak; and ~l=very weak hybridization signals;
GOLD=detection method according to Example 12;
CHEMI=chemiluminescent detection according to Example
3(b)
Applicants' hybridization results represent the
result of a subjective interpretation of the intensity of
the hybridization signals obtained after detection. DNAs
from the orqanisms listed in column two are the only ones
for which ally hybridization signal was obtained. The
panel of DNAs used for hybridization included E._coli
("E.c."), Klebsiella pneumoniae ("Kl"), Proteus vul~aris
("Pr"), Pseudornonas aer ~inosa, Sta~hylococcus
epidermatis ("SE"), Streptococcus faecalis and Homo
sapiens.
13~79~
TABLE 2
COMPARISON OE` AMES LEUl<OSTIX ASSAY
WITH APPLICANT ' S ASSAY
. ~
"LEUKOSTIX" APPLICANTS ' l1YBRIDIZATION DETECTION
RESULT RESULT SYSTEM
. .
3+ VS GOLD
3+ S CHEMI
3+ S CHEMI
3+ M CHEMI
3+ M CHEMI
3 + S GOLD
3+ S GOLD
3 + VS GOLD
3+ VS GOLD
3 + VS GOLD
3+ VS GOLD
__________________________________~____________________
2+ S CHEMI
2 + S CHEMI
2+ S (::HEMI
2 + S C HEMI
2+ S GOLD
2 + S GOLD
2+ S GOLD
2 + S GOLD
2+ S GOLD
____ ________________________________________________
1+ S GOLD
1 + VS GOLD
1 + VW GOLD
TRACE/1+ M CHEMI
TRACE VS CHEMI
TRACE W GOLD
TRACE W GOLD
TRACE VS GOLD
_____________________ _________________________________
NEG S CHEMI
NEG S CHEMI
N EG M CHEMI
NEG VW GOLD
NEG VW GOLD
NEG NEG GOLD
N E(`, NL;'G GOLD
NEG W GOLD
NEG W GOLD
NEG W GOLD
_______________________________________________________
, 51
~3~79~
~ The hybrldization results summarized in column
2 of Table 2 represerlt subjecti~e interpretations of the
in~ensity of hybridization signal obtained whe;l labeled
urine samples described in Table l were hybridized with
genomic human DNA.
The "LEUKOSTIX" assay is a colorimetric reagent
strip assay. Color development on the reagent strip is
compared to a chart provided with the assay reagent
strips and ranges frorn negative (no color development) to
3+ (very strong color development).
Example 14: Lysis of Cells
A 1.0 mL aliquot of cell suspension was
centrifuged and the cell pellet resuspended in 100/uL of
unbuffered NaOH solution. The sample was then exposed to
high temperature for a short time and then diluted to the
original volume usir,g 10 mM borate buffer. The pH of the
solution was then adjusted to neutral with HCl.
Table ~ shows the efficiency of lysis of two
different Gram positive cocci, Star~hylococcus epidermidis
and Streptococcus faecalis, at varying NaOH
concentrations at either 68C or 100'C. In this Example,
the absorbance of the 10 mL aliquots at 600 nrn was
recorded before centrifugation. After centrifugation,
the cell pellets were resuspended in varying
concentrations of NaOH (100 JuL) and duplicate samples of
each exposed to 68C for 10 minutes or 100C for 5
minutes. Each sarnple was then diluted to 1.0 mL and the
absorbance at 600 nm again recorcded. Since the beginning
and ending volumes are identical, the beginning and
ending absorbance at 600 nm provi.des a direct measurement
of lysis efficiency.
Whereas Gram negative organisms lysed
efficiently in as low as 0.1 N NaOI~, Table ~ shows
~ 3 ~ 4
clearly that ef~icient lysis is a function of both NaOH
concentration and temperature, such tha~ higher NaOH
concentra~ions are rec~uired as the lncubation temperature
decreases. ~t 100C (maximum temperature at 1
atmosphere) a concentratioll of a-t least 1.6 N NaOH was
required for efficient lysis of S. epidermidis and S.
faecalis. If lower temperatures are desirable or
necessary, then higher concentrations of NaOH will be
re~uired to maintain lysis efficiency.
TABLE 3
FFICIENCY OF LYSIS OF GRAM POSITrVE BAC~RIA
AT VARIO~S CONC~l~ATIONS OF NaOH AT 68C and 100C
-
Streptococcus faecalis
100C/5 Minutes 68C/10 Minutes
[NaOH] OD600 PREOD600 POST%LYSISOD600 P~EOD6 POST %LYSIS
0 N 0.475 0.366 23 0.512 0.357 30
0.1 .509 .261 50 .513 .238 54
0.2 .512 .194 62 .514 .259 50
0.4 .504 .175 65 .513 .150 71
0.8 .506 .113 78 .505 .147 71
1.2 .498 .082 84 .498 .150 70
1.6 .487 .061 88 .426 .099 77
Staphylocuccus epideL~idis
100C/5Minutes 68C/10 Minutes
[NaOH] OD600 PREOD600 POST%LYSISOD600 PREC)D600 POST %LYSIS
0 N 0.667 0~55S 16 0.690 0.560 19
0.1 .681 .396 42 .701 .441 37
0.2 .674 .296 60 .699 .414 41
0.4 .699 .183 74 .730 .309 58
0.8 ~705 .091 87 .715 .187 74
1.2 .680 .070 90 .719 .090 88
1.6 .693 .035 95 .660 .040 94
Example 15: Direct Labelill~ of Nucleic Acids in an
Infected Urine Sample
A set of clinical urine test samples was
collected from a hospital. 1 ml of urine was deposited
in a polypropylene tuhe (1.5 ml). To l:he urine 150 1 of
8 N-sodium hydroxide solution in wa~er was added. The
53
~L3:L~r~ ~4
mixture was maintained in a boiling water bath for five
minutes. The alkaline suspension was then transferred to
another tube containing 0.3 g of solid boric acid for
neutralization. The mixture was shaken well by hand for
mixing and solubillzation. To this mixture, 10 microgram
of the photolabelillg reagent "bio-PEG-Ang", as used in
E~ample 10 (i) was added (10 microliter of 1 mg/ml in
water). The mixture was then irradiated for 60 minutes
using a hand held UV lamp (WL25) at a long wavelength
setting. The tube was kept Oll ice during irradiation.
A nitrocellulose paper strip containing
immobilized unlabeled genomic D~A probes (as used in
Example 8), 700 microliter mixture containing 5% non-fat
dry mil~, 10% polyethylene glycol (MW-6000), lOmM sodium
pyrophosphate, all in 3 x SSC and 300 ~1 labeled test
samples were placed in a seal-a-meal bag. The bag was
heat sealed. Hybridi~ation was then conducted for two
hours at 6~C by incubating tlle sealed bag in a water
bath.
~ fter hybridization, the nitrocellulose strip
was washed at 68C with 0.1 x SSC, 0.1% ~DS for 30
minutes. The unsaturated sites were then blocked by
immersing the paper in 3% BSA solution in 0.1 mM 'crls,
0.1 M sodium chloride, 2 m~ magnesium chloride for 30
minutes. The hybrid was then de'cected by either
immunogold, or by chemiluminescence or by the BRL method
(see Examples 2, 3(a) or 3(b)). For every sample, the
diagnosis was also done by bacteriological growth
methods. The results were then compared for validity of
the present method.
It will be appreciated that the instant
specification and claims are set forth by way of
illustration and not limitation, and that various
modifications and changes may be made without departing
from thc spiri.t an~ scope of the present i.nvention.
5~
