Note: Descriptions are shown in the official language in which they were submitted.
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POLYMERIC ~;~lll)E PROBESANDUSESTHEREOF
Background of the Invention
This invention relates generally to uncharged probes and more particularly,
5 relates to the use of uncharged probes such as peptide nucleic acids (PNAs) and
morpholinos in fluorescence in situ hyhriAi7~tion assays.
.
Current diagnostic methods in infectious diseases rely upon cllltllrinp~
infectious agents from patient test samples and subsequently identifying these
infectious agents on the basis of microscopic morphology and st~ininp;
characteristics, growth requirements, etc. Under current standard growth
conditions, bacteria usually require 24 to 48 hours to grow, although some bacteria
such as Mycobacterium sp. may require weeks to grow. After snffic ient growth is~tt~inP~l, additional testing such as further bioçhemi~ tests, may be required to be
performed in order to identify the bacteria. Some of these tests require an additional
18 to 48 hours before i(lentific~tion of the bacterium can be made. ~on~;ullcllLly
with the bio~hemic~l tests, sensitivity tests may be performed in order to ~ tt~rmin~
if the bacterium is susceptible to various ~ntimicrobial agents. These methods rely
on pure cultures of each bacterium. If a mixed culture is present, additional time is
required to purify the bacteria present so that each can be identified and
susceptibility tested.
Extensive research and development of fluorescence in situ hyhri~li7~tion
(~SH) technology has occurred during the past decade. FISH routinely is applied
to gene mapping, tumor chara;L~ll;GaLion and prenatal diagnosis. See, for example,
2s J. B. Lawrence et al., Science 249:928-932 (1990); P. Lichtner et al., Science
247:64-69 (1990); E. Viegas-Pequignot et al., Proc. Natl. Acad. Sci. USA 86:582-586 (1989); B. Trask et al., Genomics 5:710-717 (1989); H. Evans et al.,
Chromosoma 48:405-426 (1974); and D. C. Tkachuk et al., Genet. Anal. Tech.
~. 8:67-74 (1991). Despite FISH's use in research and large medical center
laboratories, it is not in widespread use in clinical laboratories. Its lack of use in
clinical laboraratories probably is due to its lack of sensitivity, lack of standardized
and user-friendly protocols, long turn-around time (usually, at least about 8 hours)
and its m~nu~l, non-automated techniques, making it a labor-intensive and costlyprocedure to perform in the clinical laboratory.
3s It would be advantageous to provide an assay which would be able to detect
less than 1000 copies of target nucleic acids or a single b~t~ri~l cell in a test
CA 02221179 1997-11-14
W 096136734 PCTrUS96107075
sample. It also would be advantageous to provide a simple, rapid F~SH assay to -detect and identify b~ct~ri~l species in suspension or inside infected cells. Morover,
it would be advantageous to provide an assay for det~octing drug reci~t~n~e genes in
such b~ctPri~l species. Such detection could be performed simlllt~neously with
s iclçntific~tion. Such assays would be completed in eight hours or less and readily
~mt~n~hle to automation. It also would be advantageous to provide signal
~mrlification options such that the sensitivity of F~SH assays would be improved to
the level required to provide clinically relevant results.
o Summary of the Invention
An assay for detecting rRNA which may be present in a test sample is
provided. The assay comprises the steps of cont~ting said test sample with a
ppeptide nucleic acid (PNA) probe capable of ~ft~rhing to said rRNA in said testsample conjugated to an in~ tor reagent compri~ing signal gf n,-r~ting compound
5 capable of generating a measurable signal; and detecting said measurable signal as
an indication of the presence of rRNA in the test sample. The assay preferably is
performed by flow cytometry. Quantitation is performed by exciting fluorescence
and m~curing said signal by using a light selection filter. The signal generating
compound preferably is fluorescein or rhodamine. The rRNA in the test sample can20 be fixed prior to perforrning the assay. The assay further comprises hybric1i7ing
said test sample in situ.
The assay described hereinabove also can be performed using morpholino
compounds as probes in place of the PNAs as probes. Also, an improved
fluorescence in situ hyhritli7~tion assay is provided wherein the improvement
2s comprises hybridizing said test sample with a PNA or mo1pholino probe.
An assay for dçt~cting drug resistance genes which may be present in a test
sample also is provided herein. The assay comprises the steps of contacting saidtest sample which may contain drug resistance gene(s) with a peptide nucleic acid
(PNA) probe or a morpholino probe capable of ~tt~hing to said drug resistance
30 gene(s) in said test sample conjugated to an in-lic~t-~r reagent compri~ing signal
generating compound capable of generating a measurable signal; and ~ t~cting said
measurable signal as an indication of the presence of the drug rç~ict~nce gene(s) in
the test sample. The assay preferably is performed by flow cytometry.
Qn~ntit:~tion is p~;lrc,lllled by exciting fluorescence and m~cllring said signal by
3s using a light selection filter. The signal generating compound preferably is
fluorescein or rhodamine. The drug resistance gene(s) in the test sample can be
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fixed prior to perforrning the assay. The assay further comprises hybritli7ing said
test sample in situ.
In addition, test kits are provided for detecting the presence of rRNA or
drug re~ict~n~e gene(s) which may be present in a test sample which comprise a
s container co~ -p a PNA or morpholino probe conjugated to a signal generating
compound capable of generating a measurable signal.
Brief Description of the Drawings
FIGURE 1 presents a histogram wherein a 25 mer DNA oligo and a 15 mer
o PNA oligo probe (sequence within the 25 mer of DNA sequence) complemP-nt~ry to28S rRNA were directly labeled with fluorescein, wherein: A is a negative samplewith DNA probe labeled at both ends and B is a positive sample with DNA probe
labeled at both ends.
F~GURE 2 presents a histogram wherein a 25 mer DNA oligo and a 15 mer
15 PNA oligo probe (sequence within the 25 mer of DNA sequence~ comI~lemP.ntary to
28S rRNA were directly labeled with fluorescein, wherein: C is a negative samplewith PNA probe labeled at the amino end and and D is a positive sample with PNA
probe labeled at the amino end.
FIGURE 3 presents a photograph of stained E. coli bacteria in mouse
20 PMNs.
Detailed Description of the Invention
In situ hybridization was introduced in the late 1960's. J. G. Gall and M.
Pardue, Proc. Natl. Acad. Sci. USA 63:378-383 (1969). Generally, it involves
2s t~king morphologically intact tissues, cells or chromosomes through the nucleic
acid hybridization process to demonstrate not only the presence of a particular piece
of genetic information but also its specific location within individual cells. It does
not require the homogenization of cells and extraction of the target sequence, and
therefore, provides precise loc~li7~fion and distribution of a sequence in cell
30 populations. Secondly, the homogenization of tissues can result in a loss of
sensitivity if the target is present in only a small fraction of the cells and at a low
copy number. Such sequences would be ~liffirlllt to detect in an extract because of
a dilution effect caused by an excess of noll~a~ l nucleic acids. In situ
hybridization circumvents this problem by identifying the sequence of interest
35 concentrated in the cells c. ~ ; " i "~ it. Thirdly, if a test sample contains
heterogeneous cell populations, in situ hybridization methods can identify the type
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and the fraction of the ceIls cont~inin,~ the sequence of interest. Finally, DNA as-
well as RNA can be ~ tect~ with the same assay reagents.
FISH techniques have improved greatly during the past 15 years. The
procedure has been cimplified from its original tedious and laborious form. Withs the use of oligo probes, the assay has become more reproducible and easy to
perform. For example, this type of assay can be done wi~h multiple oligos in a one-
step protocol that can be carried out in about two hours or less (J. Bresser et al., U.
S. Patent No. 5,225,326. Attempts have also been made to automate in situ
hybridization procedures. See, for example, C. Park et al., J. Histotechnolo~y
14:219-229 (1991); E. R. Unger et al., J. Histotechnolo~y 11:253-258 (1988). Forexample, Fisher Code-On~ (available from Fisher Scienlific, Pittsburgh, PA)
series slide stainer is capable of processing 60 microscopic slides through all of the
steps required for in situ hybridization in a semi-automated fashion. In addition,
Ventana Medical Systems, Inc. (Tucson, Arizona) recently introduced a comrletely15 automated in situ hybridization system capable of h"n~lling 40 slides. For FISH
with cell suspensions and flow cytometric detection, the procedure also can be
automated.
The intrincic ~ e.Lies of in situ hybridization make it an extremely
important tool in m~fiir~,l and clinical sri~nr~s; it has great potential in the clinical
20 diagnosis of infectious diseases and cancer. Because only a small fraction
(generally less than one in a hundred to a thousand) cells in tissues harbor viruses
or bacteria, in situ hyhri~ 7tion is particularly useful in the analysis of infections.
In situ hybridization, being an anatomic method of diagnosis, complements
~7mplific~tion-based assays such as PCR and LCR which are extremely sensitive but
25 do not provide information on the cell types that are infected and qn~"~ iv~
information on the number of infected cells. In situ hyhri~li7~tion methods can
trrminP the cells which are infected, the pathogen which is infecting the cells, and
also it can determine (by qll~ntit~tive measures) the extent of the infection. Labeled
PNA probes directed against rRNA targets of microorg"nicmc offer the potential to
30 achieve the desired sensitivity and specificity with a practical protocol.
Among many potential detection schemes, fluorescence detection offers the
fastest detection technology, the ability to multiplex and the potential for good
q~l~ntit"tion in an automated format. We theorized that the combination of
fluorescence detection techniques and in situ hybridization (fluorescence in situ
35 hybridization or FISH) with so-called "peptide nucleic acids" ("PNA") probes
targeting bacterial l 6S rRNA, could be used to detect bacterial infection.
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In the search for stable antisense agents, peptide nucleic acids ("PNAs")
have emerged as useful probes for the recognition of single and double-str~n-lednucleic acids. M. Egholm et al., Nature 365:566-568 (1993). Synthetic PNA
probes are polymeric analogs of peptide. The backbone of PNA is made from
5 repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. Unlike natural
peptides which have amino acids ~tt~'h.-~ to the backbone, PNA contains purine
(A, G) and pyrimi~line (C, T) bases ~tt~-~h~l to the backbone by methylene carbonyl
linkage. Unlike DNA or other DNA analogs, PNAs do not contain any pentose
sugar moieties or phosphate groups. The molecules are neutral at physiological
o conditions. By convention, PNAs are depicted like peptides, with the N-tel.is
at the first (left) position and the C-terminn~ at the right. In addition to these
chemic~l differences, PNA is biochemically dirre~ from both peptides and
nucleic acids in that they are resistant to all known protein~cçs and nucleases. P.E.
Nielsen et al., Science 254:1497-1500 (1991); M. Egholm et al., J. Am. Chem.
Soc. 114, 1895-1897 (1992); M. Egholm et al., ibid, 114: 9677-9678 (19923; J.
Hanvey et al., Science 258: 1481-1485 (1992); P.E. Nielsen et al., Nucleic Acids.
Res 21:197-200 (1993); P. ~t~ud~ira and J. Coull, ABRF News 4(3) (1993); H.
0rum et al., Nucleic Acids Res. 21 :5332-5336 (1993). Despite these differences,PNAs have been shown to bind more strongly in solution to both DNA and RNA
than the same length DNA oligonucleotides. The use of PNAs in in situ
hybridization assays has not been demonstrated h~lc~fur~. Various factors have
contributed to the uncertainty of the use of PNAs in in situ hyhri~ tion. For
example, all cells have not only a large pool of variaty of macromolecules but also a
very complex morphological structure. A probe that can be used successfully in
2s fixed cells should have the ability to penetrate layers of cellular memberane under
suitable conditions and, at the same time, should only bind to the cle~ign~ted
target(s) but not to other components of a cell. We have discovered that
fluorescently labeled PNA probes can be used for in situ hybridization, and thatthey provide remarkably improved (i.e., six times better) signal-to-noise than the
corresponding DNA oligo. FIGURES 1 and 2 show a histogram of cell counts v.
fluorescencelOg wherein a 25 mer DNA oligonucleotide and a 15 mer PNA
oligopeptide probe (sequence within the 25 mer of DNA sequence) complt~m~nt:~,ryto 28S rRNA were directly labeled with fluorescein. The DNA oligonucleotide was
labeled at both ends, while the PNA oligopeptide was labeled only at the amino
end. When used in FISH with Chinese hamster ovary (CHO) cells and analyzed in
flow cytometry, the one fluorophore PNA probe gave three times better linear
CA 02221179 1997-11-14
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fluorescence signal to background than the two-fluorophore DNA probe. These
data are discussed in detail in the examples provided hereinbelow. The advantages
of using PNA probes are speed and the possibility of targeting short but specific
regions of b~ct~-ri~l nucleic acids. Longer pl~cmitl probes gen~or~lly require
s overnight incubation, but 30 minntes to two hours is a sufficient time for short
probes to penetrate cells and bind to the target. In addition, by carefully flt-ci nin~
the probe sequence, PNA probes can be used as a general screen of all b~cteri~l
types when the probes are complementary to a consensus region of the target (so-termed "uluvel~al probe"). On the other hand, when the probes are ~lesign~c~ to
0 hybridize to sequence specific regions of a particular bacterial species, subtyping of
bacteria can be achieved. Furthermore, the synthesis and labeling of probes are
reproducible and the labelled probes are stable for years. Also, PNA probes are
chemically very stable substitnl-ntc An additional advantage for using PNA probes
is that PNAs are resistant to all known enzymes since they are not molecules which
s exist in nature.
FISH methods targeting rRNA for the identification of microbial cells were
first introduced by DeLong et al., Science 243: 1360-1363 (1989). Due to the
ablm~l~nce of rRNA (104 and 105 copies per cell), phylogenetic ide-ntific:~tion of
microbes was demonstrated by FISH using fluorescence microscopy and
fluorescently labeled oligonucleotide probes (17-34 mers) complement~ry to
bacterial 16S rRNA. In that study, it was shown that oligo probes could
distinguish a b~ct~ rillm dubbed "son-killer" (Nasonia vitripennis) and its closest
known relative, Proteus vulgaris. These two org~ni~mc differ only slightly in size
and shape and are difficult to distinguish using standard microbiological stains.
Oligo probes labeled with two ~lirrGlGl~ fluorescent dyes easily i~ ntified one or the
other b~ terillm in a mixed culture. These researchers also demonstrated that the
rRNA content (and therefore the FISH signal) is directly proportional to the growth
rate and metabolic activities of the microorganism. Similar studies ntili7ing a flow
cytometer for fluorescence signal detection also have been docllmf ntl ~1 (R. I.Amann et al., Applied and Environmental Microbiolo~y 56: 1919- 1925 (1990); J.
G. J. Bauman et al., J. Microscopy 157:73-81 (1989); Boye and L0bner-Olesen,
The New Biolo~ist 2: 119-125 (1990); G. Wallner et al., Cytometry 14: 136-143
(1993). In these studies, i(lcntific~tion and qll~ntit~tive analysis of mixed microbial
populations were performed using fluorophore labeled oligonucleotide probes
3s specific for 11 dirr~l~l,L org~ni~m~ Flow cytometric detection of yeast by in situ
CA 02221179 1997-11-14
W 096/36734 PCTnUS96/07075
hybridization with a fluorescent rRNA probe also has been demonstrated (B. Bertin
et al., J. Microbiol. Methods 12:1-2 [1990]).
While we choose to take the advantages of in situ hykri(1i~tion, other
approaches ~or tl~t~cting and identifying microor~nicmc by targeting 16S rRNA
have been ~locum~nted. All of these methods applied DNA probes in a solution
phase hybridization format with target rRNA extracted from cells. Some of these
involved target amplification method such as polymerase chain reactions (PCR),
ligase chain reaction (LCR) or nucleic acid sequence based amplification (NASBA).
(Kohne, D. E. et al., US Patent No. 5,288,611; Greisen, K. et al., J. Clin.
Microbiol. 32:335-351, ~1994); Leong, D. U., European Patent No. EP 0 479 117
Al; Walker, G. T. et al., EP 0640 691 A2; Greisen, K. S. et al., International
Patent NO. WO 93/03186 and Lane, D. J. et al., US patent No. 5401631A).
Recently, detection and identification of microorg~nicmc inside infected
cells has been demonstrated. ~tcuhica et al., Biotechnic and Histochemistr,v
69:31-37 (1994). In this work, enzym~tic~lly labeled genomic probes were used todetect and identify Staphylococci in mouse phagocytic cells. The technique has
been extended to the diagnosis of bacteremia in the phagocytic cells of potentially
septic patient blood s~mples. ~tcllhic~ et al., Microbiol. Immunol. 38:511-517,
(1994). Current culture methods for detecting bacteria in blood samples can givefalse negative results and take two days to more than a week to obtain results,
during which time a patient may die if ~clminictered tre~tm.~nt is not dl~p~upliate.
The present invention provides an assay which has a fast turn around time and
further provides information on the specific type of bacteria present. The assaydisclosed herein also will work when more than one pathogenic species is present,
2s as is ~l~t~iled hereinbelow in the ex:~mrl~s Rapid i~ ntifi~tion of the biq~ t~-ri~l
species provides extremely valuble information for d~roL,liate patient tre~tm~ntThe assay disclosed herein (PNA-FISH) potentially can be used for
cimn1t~neoUs identification of microorg~nicmc and microbial drug recict~n~e
testing. Accompanying rapid developments in genome sequencing, more drug
resict~nre genes are being identified and isolated, which allows the development of
~ probe-based assay for direct detection of drug resistant strains at the genetic level.
For review, see A. Linton et al., SchlirL~ Ver Wasser Boden Lufthyg 78:197-224
~ (1988); J. T. C.dwrold et al., Respir. Infect. 9:62-70 (1994); and L.A. Anisimova
et al., Mol. Gen. Mikrobiol. Virusol. (USSR) 11:3-12 (1988). In this assay, a
3s hyhricli7~tion cocktail could contain probes for microbial i~lentific~tion and probes
targeting drug resistance genes or their transcripts (mRNA). Each of these probes
CA 02221179 1997-11-14
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could be :~tt~l-h.-cl to a spectrally distinct fluorescent dye to allow mnll ;1~ eter
assay on one test sample.
The present invention also encomr~Cc~s llt11i7ing a capture reagent
comprising a solid support having PNAs directly immobilized thereon which can
s can be c~nt~ t~l with a test sample, or, having the test sample directly immobilized
on the solid support. The test sample can be any liquid sllcpect~l of co~ ;.,;..g a
nucleic acid sequence which can spe~-ifi~:~lly hybridize with the immobilized
oligopeptides. The capture reagent can be contacted for a time and under conrlition.c
suitable for allowing nucleic acids in the test sample, if any, and the PNAs to
0 hybridize and thereby form hyhritli7~ti~ n complexes. The hyhri-li7~tion complexes,
if any, can be contacted with a conjugate for a time and under conditions sufficient
to enable the conjugate to specifically bind any hyhri~1i7~tion comrlext-s A signal
can then be ~l~fecte~l as an indication of the presence or amount of any nucleic acid
sequences which may be present in the test sample.
1S Immobilized PNAs as taught herein can also be employed in a "one-step"
assay configuration. According to such a configuration, a test sample suspected of
cont~ining nucleic acids which are complementary to the imrnobilized PNAs can becontacted with a conjugate for a time and under conditions suitable for allowing the
conjugate to bind any nucleic acid sequences which may be present in the test
20 sample to form conjugate/nucleic acid complexes. Alternatively, the nucleic acids
which may be present in a test sample may comprise a cletP-ct~hle moiety. Nucleic
acid sequences can be labeled or conjugated with a ~letect~hle moiety through, for
ex~mple, nick translation whereby labeled nucleotides are incorporated into a target
sequence. Conjugate/nucleic acid complexes or nucleic acids which comprise a
2s det~ct~hle moiety can then be contacted with the support bound PNAs to form
conjugate/nucleic acid/PNA complexes or nucleic acid/PNA comrleYec A signal
can then be detected as an indication of the presence or amount of any nucleic acid
sequences present in the test sample.
In another embodiment, a method for quickly detecting the presence of an
30 nucelic acids in a test sample is provided. According to this embodiment, a sample
which is suspected of cont~inin~ nucleic acids can be contacted with a support
m~tf~ri~l and the nucleic acids which may be present in the lest sample can be
immobili7P~ to the support m~tf~ri~l A conjugate can then be contacted with the
immobilized nucleic acids for a time and under conditions for allowing the
3s conjugate to bind the immobilized nucleic acids. A signal generating c~ Ju~ld
CA 02221179 1997-11-14
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compri~ing PNAs can then be detecte~l as an indication of the presence or amount of
any nucleic acids which may have been present in the test sample.
The period for which nucleic acids which are immobilized as taught herein
are contacted with, for ex~mple, a test sample, conjugate/nucleic acid complexes, or
s a conjugate is not important. However, it is ~l~;ft.led that such a contact period be
kept to a ~ illllll-l, for example, less than 30 minnt(~.s, more preferably less than 15
minutes and most preferably less than 10 minnt~s
Those skilled in the art will understand that a conjugate may comrrice a
signal generating compound capable of generating a measurable signal ~tt~t~h~l to
0 specific binding pair member . Signal generating compound (tl~tect~ble moieties)
may include any compound or conventional cletect~hle chemical group having a
~letect:~hle and measurable physical or chemical ~lup~l Ly variably referred to as a
signal. Such detectable groups can be, but are not intended to be limited to,
enzymatically active groups such as enzymes and enzyme substrates, prosthetic
15 groups or coenzymes; spin labels; fluorescent molecules such as fluorescers and
fluorogens; chromophores and chromogens; luminescent molecules such as
luminescers, chemiluminescers and biolnmin~sce~rs; phosphorescent molecules;
specifically bindable ligands such as biotin and avidin; electroactive species;
radioisotopes; toxins; drugs; haptens; polys~cch:~ricles; polypeptides; liposomes;
20 colored or fluorescent particles; colored or fluorescent microparticles; colloidal
particles such as selenium colloid or gold colloid; and the like. Additionally, a
detectable moiety can c~mprice, for example, a plurality of fluorophores
immobilized to a polymer such as that described in co-owned and co-pending U.S.
Patent Application Serial No. 08/091,149 filed on July 13, 1993, which is herein25 incorporated by reference. The ~1et~ct~hle physical or ch--mi- ~l plU~C;lly associated
with a detectable moiety can be detected visually or by an external means. Specific
binding member is a well known term and generally means a member of a binding
pair, i.e., two different mnlecllles where one of the molecules through chemical or
physical means specifically binds to the other molecule. In addition to antigen and
30 antibody specific binding pairs, other specific binding pairs inrlllcle7 but are not
intended to be limited to, avidin and biotin, antibody and hapten, complemPnt~rynucleotide sequences or complem.ontary nucleic acid sequences such as DNA or
- RNA, or PNAs, or morpholino compounds, an enzyme cofactor or substrate and
an enzyme, a peptide sequence and an antibody specific for the sequence or an
3s entire protein, dyes and protein binders, peptides and specific protein binders (e.
g., ribonuclease, S-peptide and ribonuclease S-protein), and the like. Furthermore,
CA 02221179 1997-11-14
W096/36734 - - PCTAUS96/07075
binding pairs can include members that are analogs of the original binding member,
for ex~mple7 an analyte-analog or a binding member made by recomhin~n~
techniques or molecular enginPPring- Thus, PNAs and morpholino co~ oullds are
specific binding members for DNA or RNA. If the binding member is an
s immllnnreactant it can be, for Px~mple, a monoclonal or polyclonal antibody, arecombinant protein or recomhin~nt antibody, a çhimPric antibody, a rnixture(s) or
fragment(s) of the foregoing. Signal generating compounds can be ~tt~rhpd to
specific binding pair members through any chemical means and/or physical means
that do not destroy the specii~lc binding properties of the specific binding member or
0 the ~iPt~ ct~hIe properties of the ~letPct~hle moiety.
A method provided herein can be employed to immobilize oligons to a glass
surface which is then employed in further analysis, such as a waveguide
configuration as that taught in co-owned and co-pending U.S. Patent Application
Serial No. 08/311,462 entitled "Light Sc~ttPfing Optical Waveguide Method for
15 Detecting Specific Binding Events" which is herein incorporated by reference. A
waveguide device's ability to be employed in an immunoassay type format is basedupon a phenomenon called total internal reflection (IIR). TIR operates upon tne
principle that light traveling in a denser medium (i.e. having the higher refractive
index, Nl) and stliking the intPrf~re between the denser mr~ lrn and a rarer
20 mP~ m (i.e. having the lower refractive index, N2) is totally reflected within the
denser mP~ m if it strikes the interface at an angle, qR, greater than the critical
angle, q C, where the critical angle is defined by the equa~ion:
~t C = arcsin (N2/Nl)
Under these conditions, an electr~ m~ n.-tic waveform known as an
2s "evanescent wave" is generated. The efectric field associaled with the light in the
denser mP~linm forms a st~ntling sinusoidal wave normal to the intPrfz~rc The
evanescent wave penetrates into the rarer mP-linm, but its energy E ~ ir~t~
exponentially as a function of distance Z from the intPrf~ce A parameter known as
"penetration depth" (dp) is defined as the ~ict~nre from the int~ re at which the
30 evanescent wave energy has fallen to 0.368 times the energy value at the interface.
rSee, S~lthPrl~n-l et al., J. Immunol. Meth.~ 74:253-265 (1984) ~lefining dp as the
depth where E= (e~ ]. Penetration depth is calculated as follows:
~/Nl
2~{sin2~R -(N2 /Nl)2}
Factors that tend to increase the penetration depth are increasing angle of
35 incidence, ~R; closely m~trhing indices of refraction of the two media (i.e.
_
CA 02221179 1997-11-14
W 096136734 PCTrUS96/07075
N2/Nl --> l); and increasing wavelength, ~. For example, if a quartz TIR elem~.nt
(Nl = 1.46) is placed in an aqueous mP.tlinm (N2 = 1.34), the critical angle, ~ C, is
66~ (= arcsin 0.9178). If 500 nm light impacts the int~o.rf~e at ~R = 70~ (i.e.
greater than the critical angle) the dp is a~.oxilllately 270 nm.
s TIR has also been used in conjunction with light sc~tte.ring detection in a
technique referred to as Scattered Total Tnt~.rn~l P~flect~n~e ("STIR"). See, e.g.,
U.S. Patents 4,979,821 and 5,017,009 to Schutt, et al and WO 94/00763.
According to this technique, a beam of light is sc~nn~-~l across the surface of a TIR
element at a suitable angle and the light energy is totally reflecte.cl except for the
evanescent wave. Particles such as red blood cells, colloidal gold or latex
specifically bound within the penetration depth will scatter the light and the scattered
light is (ie.tectecl by a photodetection means.
Immobilizing oligos contained to support m~tt~.ri~l.c according to the present
invention comprises contacting a support m~t~.ri~l with an oligo solution and drying
the solution upon the support m~t.-.ri~l. If the oligo is suspected of being contained
within a test sample, the test sample is dried upon the support m~te.ri~l.
Support m~t~.ri~ or solid ~uppolL~ (so-termed "solid phases") to which
oligos can be immobilized are well known in the art and include m~t~ri~l~ that are
subst~nti:~lly insoluble. Porous m~teri~l.c can serve as solid supports and may
include, for example, paper; nylon; and cellulose as well as its derivatives such as
nitrocellulose. Smooth polymeric and nonpolymeric m~t~.ri:~l.c are also suitablesupport m~te.ri~l~ and in~.ln~le., but are not intPn-lecl to be limited to, plastics and
derivatized plastics such as, for example, polycarbonate, poly~lyl~lle~ and
polypropylene; magnetic or non-m~gnt~tir metal; quartz and glass. Preferably,
quartz, glass or nitrocellulose is employed as a support m~t~-ri~l Solid supports
can be used in many configurations well known to those skilled in the art
including, but not limited to, test tubes, microtiter wells, sheets, films, strips,
beads, microparticles, chips, slides, cover slips, and the like.
Oligonucleotides according to the invention will be understood to mean a
sequence of DNA or RNA, whereas the term oligopeptides will be understood to
mean a sequence of PNA or morpholino compounds. All may be generally terrned
as oligos herein. Both PNAs and morpholino compounds have a higher binding
affinity, better penetrability and lower susceptibilityt o enzymatic digestion than
nucleic acid probes. The length of an oligo which is immobilized to a support
3s m~t.o.ri~l is largely a matter of choice for one skilled in the art and is typically based
upon the length of a complementary sequence of, for example, DNA, RNA, or
CA 02221179 1997-11-14
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PNA or morpholino compound which will be captured. While the length of an
immobilized oligo is typically between about 5 and about 50 base pairs, preferably,
the length of an immobilized oligo is between about 5 and about 30 base pairs,
more typically between about 10 and about 25 base pairs.
s A "capture reagent", as used herein, refers to an unlabeled specific binding
member which is specific either for the analyte as in a sandwich assay, for the
inclic~t-~r reagent or analyte as in a com~cliLive assay, or for an ancillary specific
binding member, which itself is specific for the analyte, as in an indirect assay.
The capture reagent can be directly or indirectly bound to a solid phase m~trri~l
before the performance of the assay or during the perf~rm~nre of the assay, thereby
enabling the separation of immobilized complexes from the test sample .
Test samples which can be tested by the methods of the present invention
described herein include human and animal body fluids which can contain nucleic
acids such as whole blood, serum, plasma, cerebrospinal fluid, urine, biologicalfluids such as cell culture supern~t~nt~ fixed tissue specimens and fixed cell
specimens. It also is within the scope of the present invention that a variety of non-
human or non-animal body fluids which can contain nucleic acids also can be
analyzed according to the present invention.
Synthesis of oligos is routine using automated synthesizers. If desired,
automated synthr~i7~ rs can produce oligoss which are modified with terminal
amines or other groups. A useful review of coupling chemistries is found in
Goodchild, Bioconju~ate Chemistry. 1(3):165-187 (1990).
The amount of oligo solution (which can be a test sample) that is applied or
"spotted" upon a solid support need be large enough only to capture sufficient
2s complementary sequences to enable detection of, for çx~mple, a captured sequence
or conjugate. This is dependent in part on the density of support m~t.-ri~l to which
the capture oligo is immobilized. For example, areas of as little as 150 ,um in
m~ter may be employed. Such small areas are preferred when many sites on a
support material are spotted with oligonucleotide solution(s). The practical lower
limit of size is about l~Lm in diameter. For visual detection, areas large enough to
be detrct.o~1 without m~gnifir~tion are desired; for example at least about 1 to about
50 mm2; up to as large as 1 cm2 or even larger. There is no upper size limit except
as t~ t~te(l by m~nnf~rtllrin~ costs and user convenience.
Once an oligo solution is contacted with a solid support, evaporation is the
3s pler~llt;d drying method and may be performed at room te~ Lu.c (about 25~C).When desired, the evaporation may be performed at an elevated temperature, so
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long as the l~ ul~ does not cignific~ntly inhibit the ability of the oligos to
specifically hybridize with complement~ry sequences.
The process of immobilizing oligos to a solid support may further c~ mprice
"baking" the support m~teri~l and the oligo solution thereon. Baking may includesubjecting the solid phase and oligonucleotide solution residue, to temperaturesbetween about 60~C and about 95~C, preferably between about 70~C and about
80~C. The baking time is not critical and preferably lasts for between about 15
minlltes and about 90 minllt~C Baking is particularly ~rert;llc;d when porous
support m~teri~lc such as, for example, nitrocellulose are employed.
0 An overcoating step may optionally be employed in the method herein
provided. Overcoating typically comprises treating the support m~t~ri~l so as toblock non-specific interactions between the support m~t~ri~l and complem~ntz~ry
sequences which may be in a fluid sample. It is pl~rellt;d that the overcoating or
blocking m~t.-ri~l is applied after the oligo solution has been dried upon the support
m~ttori~l In cases where a baking step is employed, the blocking m~teri~l should be
applied after the baking step. Suitable blocking m~t~-ri~lc are casein, zein, bovine
serum albumin (BSA~, 0.5% sodiumdodecyl sulfate (SDS), and lX to SX
Denhardt's solution (lX Denhardt's is 0.02% Ficoll, 0.02% polyvinylpyrrolidone
and 0.2 mg/ml BSA). Other blockers can include del~ elll~ and long-chain water
soluble polymers.
Casein has been found to be a ~lertll~d blocking m~tt-ri~l and is available
from Sigma Chemical, St. Louis, MO. Casein belongs to a class of proteins
known as "meta-soluble" proteins (see, e.g., U.S. Patent 5,120,643 to Ching, et
al, incorporated herein by reference) which are preferably treated to render them
2s more soluble. Such treatm~ntC include acid or ~lk~line tre~tm--nt and are believed to
perform cleavage and/or partial hydrolysis of the intact protein. Casein is a milk
protein having a molecular weight of about 23,600 (bovine beta-casein), but as
used herein, "casein" or "alkaline treated" casein both refer to a partially hydrolyzed
mixture that results from ~lk~linl- tre~tm~nt as descrihed in ex~mple 1 of US Patent
5,120,643. An electrophoresis gel (20% polyacrylamide TBE) of the so-treated
casein shows a mixture of fragments predominantly having molecular weight less
than 15,000, as shown by a diffused band below this marker.
- A ~lc;r~ d assay method for the detection of nucleic acids in a test sample
according to the present invention includes flow cytometric procedures and particle
counting procedures. For example, in particle counting, analytes which are
members of specific binding pairs are quantified by mixing an aliquot of test sample
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with microparticles coated with a capture reagent capable of binding to the nucleic
acid of interest as the other member of the specific binding pair. If the nucleic acid
is present in the test sample, it will bind to some of the microparticles coated with
the capture reagent and ag~ t~s will form. The analyte concentration is
s inversely proportional to the nn~ tin~t~d particle count. See, for example, Rose
et al., eds., Manual of Clinical Laboratory Immunolo~. 3rd edition, Chapter 8,
pages 43-48, American Society for Microbiology, Washington, D. C. (1986).
Flow cytometry methods that sense electronic and optical signals from cells
which are illnTnin~t~d allows deteTminz~tion of cell surface char~rt~Ticti~s, volume
0 and cell size. Nucleic acids present in, for ex~mrle7 bacteria present in a test
sample are bound to the PNA or comorpholino compound and detected with a
fluorescent dye which is either directly conjugated to the PNA or morpholino
compound or added via a second reaction. Different dyes, which may be excitable
at dirr~lt;nt wavelengths, can be used with more than one PNA or morpholino
15 compound specific to dirr~ L nucleic acids such that more than one type of nucleic
acid can be detected from one sample. In fluorescence flow cytometry, a
suspension of particles, typically cells in a test sample, is transported through a
flowcell where the individual particles in the sample are illnmin~t~-d with one or
more focused light beams. One or more detectors detect the illl~;l,~;Lion between the
20 light beam(s) and the labeled particles flowing through the flowcell. Commonly,
some of the dett~-ct- rs are decign.-~l to measure fluorescence emissions, while other
detectors measure scatter intensity or pulse duration. Thus, each particle that passes
through the flowcell can be mapped into a feature space whose axes are the
emission colors, light intensities, or other plop~llies, i.e., scatter, measured by the
2s detectors. In one situation, the dirr~l~ nt particles in the sample ~nap into distinct
and non-overlapping regions of the feature space, allowing each particle to be
analyzed based on its mapping in the feature space. To prepare a test sample forflow cytometry analysis, the Op~ldtc)l m~nn~lly pipettes a volume of test samplefrom the sample tube into an analysis tube. A volume of the desired fluorochrome30 labeled PNA or morpholino compound is added. The sample/PNA or morpholino
compound mixure then is incubated for a time and under conditions sllfflcient toallow nucleic acid/PNA or morpholino compound bindings to take place. After
incubation, and if necessary, the operator adds a volume of RNS Iyse to destroy
any RBCs in the sample. After lysis, the sample is centrifuged and washed to
3s remove any left-over debris from the lysing step. The centrifuge/wash step may be
repeated several times. The sample is resuspended in a volume of a fixative and the
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sample then passes through the fluorescence flow cytometry instrument. A method
and a~aldLus for performing flow ~lltom~t~-~l analysis is ~lescrihe l in co-owned
U.S. Patent application Serial No. 08/283,379, which is incorporated herein by
reference. It is within the scope of the present invention that microspheres can be
s utilized in the methods desçrihe~ herein, tagged or labeled, and employed for in
vitro diagnostic applications. It also is within the scope of the present invention that
other cells or particles, including bacteria, viruses, durocytes, etc., can be tagged or
labeled with the PNAs or morpholino compound as described by the present
invention and used in flow cytometric methods.
It is c.~llLt;~ lated that the reagent employed for the in vitro assays can be
provided in the form of a kit with one or more cont~inerc such as vials or bottles,
with each container col-t~i.li-.g a separate reagent such as a PNA or morpholinocompound, or a cocktail of these compounds, employed in the assay(s). These kitsalso could contain vials or cont~inerC of other reagents needed for performing the
assay(s), such as washing, processing and intlic~tor reagents.
The present invention will now be described by way of examples, which are
meant to illustrate, but not to limit, the spirit and scope of the invention.
EXAMPLES
For convenience only, the Sequence Listing provided herein contains a
listing of both DNA and PNA probes. The PNA sequences in the Sequence
T icting, denoted as SEQUENCE I.D. NOS. 4 through 9, are stated in the Sequence
Listing as having fluorescein labelling at the 5' end of the m~lecule7 and are denoted
as DNA. Actually, the amino-termin~l end of the PNA mnl~c7lle was labelled with
2s fluorescein. Also, the molecule in SEQTJENCE I.D. NOS. 4 through 9 are PNA
molecules, and not DNA moleculès. The arnino end of the PNA molecule was
termed the 5' end in the Sequence Listing but is not considered as the 5' end of an
oligopeptide. A PNA molecule is not a DNA genomic molecule.
Example l. Comparison of FISH and DNA Probe Hybridization Efficiency
A. EA~e~ lellLal Protocol. PNA and DNA probe hybridization efficiencies were
compared using protocols and hybridization conditions o~Li~ d for DNA.
Detection was by an EPICS(~)Profile II (Coulter Corp., Hialeah, Florida) flow
3s cytometer equipped with an Argon laser. The laser was set to 15mW at 488nm.
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16
Fluorescein fluorescence was acquired using the light selection filter of 525/30 nm
(central wavelength/full bandwidth at half-m~xim~l trz~ncmiccion).
In this t;~.; " ,Pnt the flourescent signal intensity of the PNA
(SEQUENCE I.D. NO. 4) and DNA probes (SEQUENCE I.D. NO. 1) were
s compared. The DNA oligo probe (SEQUENCE I.D. NO. 1) was a 25mer that
amplified 28S rRNA of m~mm~ n cells. It was labeled with fluorescein at both
the 3' and 5' ends. The PNA probe (SEQUENCE I.D. NO. 4) was a l5mer whose
sequence resided within that of (SEQUENOE I.D. NO. 1). SEQUENCE I.D. NO.
1 was labeled with fluorescein only at the amino-end of the polypeptide. The
10 negative control DNA probe (SEQUENCE I.D. NO. 2) was a 25mer
complimPnt~ry to pBR322 sequence and labeled in the same manner as
SEQUENCE I.D. NO. 1. The negative control PNA probe (SEQUENCE I.D.
NO. 5) was a l5mer complim~nt~ry to Hepatitis B viral DNA (positions 330-344).
It was labeled in the same manner as SEQUENCE I.D. NO. 4.
B. Cell Fixation. Chinese Hamster Ovary (CHO) cells ~A.T.C.C. No. CRL 9618,
available from the American Type Culture Collection [A..T.C.C.], 12301 Parklawn
Drive, Rockville, MD 20852) were grown in F12 mP(1ium, supplt-mPnted with
10% serum (Cat. No. 16140, Life Technology, Grand Island, NY). The cells were
collected by trypsinization and centrifugation at 450 x g for 10 min (IECCentra-8R
Centrifuge, International Equipment Co., Nee~lh:~m Hts., MA). The cells were
washed with PBS (0.14 M NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM
KH2P04, pH 7.4) ~wice and fixed immP~ tPly with 4% paraformaldehyde for 15
min at room te~ elalul~. The cells then were washed twice with PBS and stored
2s either in PBS at 4~C for up to a week or in 70% ethanol for months at -20~C.
C. In Situ Hvbridization. Cells were incubated in 0.01% pepsin in 0.02 N HCl for10 min at 37~C, washed once with PBS + 0.02% glycine for 2 min and finally
washed twice more with PBS. The cells were post-fixed with 2%
paraformaldehyde for 5 min at room Lemperature, washed with PBS once and then
resuspended in HBSS (0.14 M NaCl, 5.4 mM KCl, 0.7 rnM NaHPO4, 1 mM
NaHCO3, pH 7.3). One volume of 20X SSC (3 M NaCl, 0.3 M sodium citrate,
pH 7.0) and 2 volumes of fn" ~ 1P were added to the cells, and then they were
prehybridized at room temperature for at least 10min. Meanwhile, probes (20 pmolPNA [SEQUENCE I.D. NO. 4 ] or DNA [SEQUENCE I.D. NO. 1] per million
cells) were resuspended in 15 ~11 hybridization mixture (2X SSC, 50% f~rm~
0.5% SDS, 100 ~glml Salmon sperm DNA [available from Boehringer Mannhein,
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Tn~ n~polis, IN) to form a probe mixture. The probe ~ Lule was heated in a -
boiling water bath for 2 min and cooled down rapidly on ice. Cells were spun
down at 450 x g for 10 min and the probe mixture was hybridized to the cells.
Probes then were hybridized for 3 hours at 40~C. The cells were washed first with
s 2 X SSC, 50% form~mide and 0.5% SDS, then 2X SSC with 0. lX SSC, each for
30 min at 40~C. The cells which were resuspended in PBS were analyzed by flow
cytometry.
D. Results. Stained cells were analyzed by flow cytometry (EPICS(~)Profile II,
lo Coulter Corp., Hialeah, Florida), equipped with an Argon laser. Average linear
fluorescence signals of the negative control cells and the positive samples are listed
below in TABLE 1. The results clearly showed that the PNA probe (SEQUENCE
I.D. NO. 4 ), although much shorter in length, provided a hi~her signal to noiseratio compared to the corresponding DNA probe (SEQUENCE I.D. NO. 1 ). The
PNA probe (SEQUENCE I.D. NO. 4) provided approximately six times higher
signal to noise ratio, conci(lering that the PNA probe (SEQUENCE I.D. NO. 4 )
had only one fluorescent label as compared to the DNA probe (SEQUENCE I.D.
NO. 1 ) which had two.
'rABLE 1
ProbeType Neg. Ctrl. Probe 28S rRNAProbe Signal toNoise
DNA 25mer-2FL 0.209 3.295 15.8
(SEQ I.D. NO. 1) (SEQ I.D. NO.2.) (SEQ I.D. NO.1 )
PNA l5mer-lFL 0.331 15.18 45.9
(SEQ I.D. NO. 4) (SEQ I.D. NO.5 ) (SEQ I.D. NO. 4)
Example 2. Ionic Strength Comparison
25 A. Experimental Protocol. In this experiment, different salt buffers in the
hybridization cocktail were tested using CHO cells as clesçrihe~l in Example 1. The
FISH protocol as described in Example 1 was followed. Twenty (20) pmol of the
EuB338 probe (SEQUENCE I.D. No.7 ) was dissolved per cell sample in various
~ hybridization cocktails for c~-mp~n~on. A negative control sample (treated with 100
30 ~Lg/ml RNase A [available from Sigma, St. Louis, MO] in PBS at room tt~ dLu
for 1 hour) was included for each hybridization cocktail tested.
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18
The compositions of hyhric1i~tion cocktails culllL,alc;d were (1) B 1: 2X
SSC,50% formaTnide (Molecular Biology Grade, available from Fisher Scientific,
Pittsburgh, PA),0.5% SDS (available from Sigma, St. Louis, MO) and 0.1 % BSA
(available from Sigma, St. Louis, MO); (2) B2: PBS,50% forrn~mi~le, O.5% SDS
S and 0.1% BSA; (3) B3: TE (10 mM Tris, l mM EDTA),50% foTTn~mi~le~ 05%
SDS and 0.1% BSA.
B. Results. The stained s~mples were analyzed by flow cytometry as described in
Example 1. Average fluorescence intensity of negative control and positive samples
lo are shown in TABLE 2. The signal to noise ratio was defined as the fluorescence
intensity of positive sample divided by that of the corresponding negative control.
TABLE 2
Fluoresc. Intensity Fluoresc. Intensity
Samples of Neg. Control of Pos. Sample Signal toNoise
B 1 0.306 5.064 16.3
B2 0.403 6.555 16.4
B3 0.982 6.501 6.8
The results as shown above in TABLE 2 demonstrate that the fluorescence
signals of PNA hybridization to the target RNA was not dependent on the salt
buffer used in the hybridization cocktail. In TE (lOmM Tris.Cl, lmM EDTA, pH
7.4) buffer, however, nonspecific binding of the probe to the target was much
20 greater than that in SSC or PBS buffer. This was reflected in a reduced signal to
noise ratio. However, PBS and SSC buffers had equiva]ent signal to noise of
about 16. The SSC buffer is commonly used in fluorescence in situ hybridization
with DNA probes.
2s Example 3. Evaluation of Denaturing ]~ea~ents
This example was designed to fl~terrnine the optimal concentration of
den~ rin~ reagents for PNA in situ hybridization.
A. Cell Fixation. Briefly, E. coli (available from the A.T.C.C., 12301 Parklawn
Drive, Rockville, MD 20852 as ATCC deposit number 8739) was grown in
trypticase soy broth (TSB, available from DIFCO Laboratories, Detroit, MI) at
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35~C overnight. Then, the cells were ~lxed with 4% paraf ~ yde for 15 min
at room l~ -dLul~. The cells next were washed with PBS twice and stored in
PBS at 4~C for up to a week.
~ .
s B. In Situ Hybridization. E. coli cells in PBS ([A.T.C.C. deposit number 8739]lml per sample, 109 cells/ml), prepared as described in this F.x~mple hereinabove,
were pelleted by centrifugation at 1000 x g for 10 min. The cells were resuspended
in 20 mM Tris + 2 mM CaC12. Proteinase K was added to 1 ~Ig/ml and incubated
at 37~C for 7.5 min. The cells then were washed with PBS + 0.02% glycine,
0 followed by two more washes with PBS. For negative control samples, E. coli
cells in PBS were incubated with 100 ,ug/ml RNase A for 1 hour at room
temperature. The cells then were washed twice with PBS. All samples were spun
down and resuspended in 100 ,ul of hybridization cocktail without probe and
allowed to pre-hybridize for 10 min at room temperature. The cells next were
15 pelleted, and 20 ,ul hybridization cocktail with 20 pmol of Probe 7 (SEQUENCEI.D. NO. 7 ) was added to the cell pellet and allowed to hybridize for 3 hours at
38~C.
Hybridization cocktails with 50% fo. " .~ i(le (standard for DNA
hybridization), various concentrations of urea or no den~ ring agents were tested
20 by the methods described herein. The compositions of various hyhri~ ti~ n
cocktails are shown in TABLE 3.
TABLE 3
BUFFER DESIGNATION COMPOSITION
B0 PBS+10% DMSO (Sigma) + 0.5%SDS +
0.1% BSA --- NO DENATURANT
B1 PBS + 10% DMSO + 0.5% SDS + 0.1%
BSA + 50% foramide
B2 PBS + 10% DMSO + 0.5% SDS+ 0.1%
BSA + 0.25M urea
B3 PBS + 10% DMSO + 0.5% SDS + 0.1%
BSA + 0.5M urea
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TABLE 3 (CONTrNUED)
BUFFER DESIGNATION COMPOSlIION
B4PBS + 10% DMSO + 0.5% SDS +
0.1 %BSA + lM urea
B5PBS + 10% DMSO + 0.5% SDS + 0.1%
BSA+2Murea
B6PBS + 10% DMSO + 0.5% SDS + 0.1%
BSA + 4M urea
B7PBS + 10% DMSO + 0.5% SDS +
0.1%BSA + 8M urea
C. Results. After hybridization, cells were analyzed on the same flow cytometer
described in the previous examples. The average fluorescence int~,ncities of negative
controls and positive samples were recorded and are presented in TABLE 4. Signal5 to noise (S/N) were calculated as described previously in Example 2.
TABLE 4
Fluores. Tnt.-ncify of Fluoresc Tnte,ncity of
S~rnplecNeg. ControlPos. SampleSignal to Noise
B0 0.108 1.365 12.6
Bl 0.107 1.347 12.6
B2 0.107 1.159 10.8
B3 0.109 1.127 10.3
B4 0.107 1.238 11.6
B5 0.109 1.083 9.9
B6 0.111 0.867 7.8
B7 0.123 1.078 8.8
The results shown above in TABLE 4 demonstrate that the PNA
hybridization signal was not affected by cl~ n~tnring agents such as 50% fonn:~n~
This hybridization is commonly used for DNA probe in silu hyhri~li7~ti-~n
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However, urea, especially at high concentrations, we~kt-neA the hybridization
between the PNA probe (SEQUENCE I.D. NO. 7) and the RNA target.
As is known in the art, the purpose of using ~len~hlring agent in DNA probe
hybritli~tionc is to make the target ~rcçscihle to the probe. The targets, double
stranded or single stranded but with complicated secondary structure, have low
hybridization efficiencies if den~t~lring agents such as f~rm~mitle are not used.
PNAs, however, have a much stronger binding efficiency to the target and can
unwind double stranded DNA upon strand-~licpl~rement (Egholm, M. et al., J.
Am. Chem. Soc. 114:9677-9678, (1992); Cherny, D. Y. el al., Proc. Natl. Acad.
0 Sci. U. S. A. 90:1667, (1993)). These data demonstrate that rl~rl~tllring agents for
Villg probe ~rCçccihility can be elimin~to~l when utilizng PNAs, as a
comparison of hybridization signal to noise ratios (S/N) of sample B0 and B 1
~TABLE 4) clearly demonstrate. It thus was not surprising that the hyhri~ tion
signal decreased as the conrçntr~tion of ~len~t lrinp agent increased, since
clen~tllring agent such as urea can disrupt hydrogen bonds, the primary force for the
hybridization of PNA to DNA and RNA.
Example 4. Effect of Other Components in the Hybridization Buffer
A. Experimental Protocol: In this experim.ont, hyhri(li7~tion buffer conditions were
optill~ d and simplified for DNA probes. Conditions tested included a permeationreagent (DMSO), a detergent (SDS), a non-specific binding blocking reagents
(BSA and Salmon sperm DNA). The following buffer conpositions used are
presented in TABLE 5.
TABLE 5
B~FFER DESIGNATION COMPOSlTION
Bl PBS
B2 PBS +10% DMSO
B3 PBS + 0.5% SDS
B4 PBS + 10% DMSO + 0.5% SDS
B5 PBS + 10% DMSO + 0.5% SDS + 0.1%
BSA
B6 PBS + 10% DMSO + 0.5% SDS + 0.1%
BSA + 100ug/ml Salmon sperm DNA
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Eub338 probe (SEQUENCE I. D. NO 7), dissolved in diLrclcllL
compositions described in TABLE 5 was added to E. coli and the negative controls(E. coli treated with RNase). After hyhri~i7~tion as described in Experiment 3,
samples were analyzed on flow cytometer.
B. Results:
The results of the above-described experiment are sllmm~ri7~1 below in
TABLE 6.
TABLE 6
Fluorescence ofFluorescence of Signal-to-
BufferNeg. Control positivesample Noise
Bl 0.109 0.124 1.14
B2 0.107 0.127 1.19
B3 0.106 1.273 11.98
B4 0.106 1.315 12.41
B5 0.106 1.085 10.23
B6 0.106 0.808 7.62
The data from TABLE 6 shows that SDS is a critical factor in the
hybridization. Other commonly used components in a typical DNA-FISH assay,
such as DMSO and BSA, did not .signifi~ntly affect the hybridization. Salmon
5 sperm DNA is used in DNA-FISH hyhritli7~tion cocktails to block positively
charged cellular components. Since PNAs are neutral, it was hypoth~,si7~cl that
such blocking of DNA may not be needed in the hybritli7~fi- n cocktail for PNAs.Our data snmm:~ri7~-~l hereinabove in TABLE 6 confirm~1 this hypothesis.
Example 5. Effect of Dctc~ L~ in Hybridization Buffer
A. Experimental Protocol. This c~c~ ent compares the effect of dirr~cnL ~-
dc~e~cnL~ in the hybridization buffer. E. coli cells were grown, fixed and
hybridized to Eub338 probe (Sequence I.D. NO.7) as described in Example 3. The
2s compositions of various hybridization cocktails are shown in TABLE 7.
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TABLE 7
BUFFER DESIGNATION COMPOSl~ION
B 1 PBS+0.1 %SDS
B2 PBS + 0.5% SDS
B3 PBS + 0.1% Tritor. X-100
B4 PBS + 0.5% Triton X-100
B5 PBS + 0.1 % Tween-20
B6 PBS + 0.5% Tween-20
B. Results:
After hybridization, the cells were analyzed on the same flow cytometer.
The average fluorescence i~lr~cili~s of negative controls and positive samples were
recorded and are presented in TABLE 8. Signal to noise (S/N) was calculated as
described previously in Example 2).
TABLE 8
Fluores.Intensity of Fluoresc.Intensity of
SamplesNeg. ControlPos. SampleSignaitoNoise
Bl 0.106 1.975 18.6
B2 0.131 2.118 16.2
B3 0.112 0.263 2.34
B4 0.131 0.301 2.3
B5 0Ø110 0.268 2.44
B6 0.116 0.266 2.29
The data presented in TABLE 8 clearly shows that the PNA probe did not
bind efficiently to the target rRNA in the presence of commonly used detergents in
DNA-FISH assays such as Triton X-100(~) or Tween-20@~). Thus, SDS must be
included in the hybridization buffer to provide probe ~f~ceccihility.
Example 6. Application of PNA FISH Hybridization to Alternative Targets
A. Experimental Protocol. In this experiment, two types of bacteria (two strains of
the yeast Saccharomyces Cerevisiae (strain YJO, obtained from Dr. B. Kohorn,
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Dept. of Botany, Duke University, Durham, NC., PNAS 88:5159-5162, ~1991]
and strain E8-1 lC, obtained from Dr. E. T. Young, Univ. of W~chington, Seattle,WA), and a gram-positive bacteria Staphylococcus aureus, ATCC 6538, obtained
from the A.T.C.C., 12301 Parklawn Drive, Rockville, Maryland 20852) were
5 hybridized to the 16S rRNA universal probe (SEQUENCE I.D. NO. 6 ), as
follows, to demonstrate the universality of this PNA FISlH approach. RNase
treated samples were used as negative controls.
Yeast cells were grown in yeast peptone dextrose (YPD, DIFCO
Labolalolics, Detroit, MI) m~ ium overright at 29~C and S. aureus was grown in
10 trypticase soy broth m,-~1ium (TSB) (DIFCO Laboratoreis7 Detroit, MI) overnight at
35~C. The cells were collected by centrifugation at 2500 Ipm for 8 min and washed
with PBS three times. Cells were fixed and treated as described in Example 3. One
(1) x 108 cells were used in each sample. Twenty (20) pmol of the universal probe
(SEQUENCE I.D. NO. 6 ) which hybridized to all 16S like rRNA was dissolved in
hybridization buffer (PBS + 0.5% SDS), applied to each sample (108 cells) and
allowed to hybridize. After post-hybridization washes with PBS, the cells were
analyzed as described in Fx~mple 3.
B. Results. The results were sllmm~ri~l in TABLE 9. The data in TABLE 920 demonstrated that the PNA in situ hybridization protocol developed in this study
can be generalized to a gram-positive b~ct~rillm and yeast .
TABLE 9
Fluoresc. Intensity. Fluoresc. Intensity Signal to
Samples of Neg. Control of Pos. Sample Noise
S. cerevisiae YJO 0.106 0.963 9.08
S. cerevisiae E8- l l C 0.322 2.118 6.58
S. aureus 0.109 0.39 3.58
Example 7. PNA Probe Specificity
A. Experimental Protocol. This experiment tested the specificity of the PNA in situ
hybridization protocol developed in Example 1. Five (5) ~lirr~ probes were
30 applied to E. coli. (ATCC deposit number 8739, obtained as described
hereinabove). The simplified and optimized protocol developed for PNA and
CA 02221179 1997-11-14
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described in Example 3 was followed. TABLE 10 presents the probes used in the
study.
TABLE 10
PROBE DESCRIPIION
Probe 4 (SEQUENCE I.D. No. 4) complementary to m:~mm~ n 28S rRNA
(position 1901-1915)
Probe 9 (SEQUENCE I.D. No. 9) complem~nt~ry to S. Aureus 16S rRNA
found only in (position 78-93)
Probe 6 (SEQUENCE I.D. No. 6) complementary to the conserved region of
microbial 16S rRNA (position 1392-1406)
Probe 7 (SEQUENCE I.D. No. 7) complementary to conserved region of
eubacterial 16S rRNA (338-352)
Probe 8 (SEQUENCE I.D. No. 8) complementary to conserved region of
eukaryotic 16S rRNA (position 1209-1223)
B. Results. E. coli cells were stained with the five different probes described
hereinabove and analyzed by flow cytometry as described in Example 1. The
results are shown in TABLE 11.
TABLE 11
Fluoresc. IntensityFluoresc. Intensity Signalto
Probes Used of Neg. Control of Pos. Sample Noise
Probe 4 0.105 0.182 N/A
(SEQ I.D. No. 4)
Probe 8 0.107 0.107 N/A
(SEQ I.D. No. 8)
Probe 9 0.105 0.110 N/A
(SEQ I.D. No. 9)
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26
TABLE 11 (Cont.)
Fluoresc. IntensityFluoresc. Intensity Signalto
Probes Usedof Neg. Controlof Pos. Sample Noise
Probe 6 0.105 0.726 6.9
(SEQ I.D. No. 6)
Probe 7 0.106 1.074 ~ 10. 1
(SEQ I.D. No. 7)
The results shown in TABLE 11 clearly illustrate the specificit~ of the
hybridization technique when used with PNA probes. The m~mm~ n 28S rRNA
probe and S. aureus probe did not cross-react to E coli, while the 16S rRNA
5 universal probe and the eubacterial probe hybri(li7~cl as expected to ~. coli.
Example 8. Comparison of DNA and PNA Probes Under Conditions
Preferred by Each
0 A. Experimental Protocol. This experiment co~ al~;d DNA and PNA probes in a
E. coli system under both conditions optimized for DNA F~SH and PNA FISH..
The EuB338 sequence was used to synth~si7f~ a DNA 18mer (SEQUENCE I.D.
NO. 1 ) and a PNA 15mer (SEQUENCE I.D. NO. 7 ). Both the SSC buffer
system and experimental protocol optimized for DNA probes (Example 1) and the
15 PBS buffer system and experimental protocol opl illfi~ed for PNA probe (F.x~mples
3-5) were tested. The optimi7~3ri PNA-FISH buffer system was composed of PBS
and 0.5% SDS. The ~L)Lhlli~d SSC buffer system (which is conventional for
DNA) was composed of 2X SSC, 50% formamide, 0.5% SDS and 100 ~g/ml
salmon sperm DNA.
B. Results. The data shown in TABLE 12 clearly demonstrate that the PNA probe
(SEQUENCE ID NO. 7 ), whether in PBS or SSC buffer (conventional FISH
buffer system), provided signifi~ ~nt ~iiccrimin~tion (signal-to-noise). Also, the
signal was better in the o~ ed PBS buffer. The corresponding DNA probe
25 (SEQUENCE ID NO. 3), however, showed ~i nific~ntly less (about 6-10 fold)
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signal to noise. The DNA probe (SEQUENCE ID NO.3) also performed better in
the SSC buffer cu~ ;llg f~rm~midi as a dprl~hlring agent, which was exrecteA
TABLE 12
Fluoresc. Intensity Fluoresc. Intensity Signalto
Samples of Neg.control of Pos.Sample Noise
Probe 7 0.108 2.756 25.5
(SEQUENCE ID
NO. 7)
PNA-PBS buffer
Probe 7 0.131 1.964 15.0
(SEQ ID ~7)
PNA-SSC buffer
Probe 3 0.106 0.198 1.87
(SEQ ID #3)
DNA-PBS buffer
Probe 3 0.110 0.250 2.27
(SEQ ID ~3)
DNA-SSC buffer
Example 9. Detectm~ Bacteria inside Phygocytes
Images were taken using Metamorph software package (Universal Tm~ging
10 Corporation, West Chester, PA) and Nikon epi-fluorescence microscope equippedwith a mercury arc lamp and a cooled Star I CCD camera (Photomt~ s, Tucson,
Arizona). The light source used was an HBO lOOw HG mercury bulb (Fryer Co.,
Inc., Huntley, IL). Images,576 x 384 pixels, were taken using a Nikon lOOX oil
immt-rcion objective.
A. In vitro infection. Three (3) ml of 6% dextran was added to 10 ml of human
blood obtained from a healthy donor and incubated at 37~C for 30 min. The PMN-
rich layer then was collected. It was incubated with a preparation of E. coli
previously prepared by incubating 10 ml of an E. coli solution (having an
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W096/36734 PCTrUS96/07075
28
absorbance of 0.1 at 600 nm wavelength, in~llk~tecl for 30 min at 37~C, having the
sllp~rn~t~nt ~le~nttoA and using the rem~ining layer of b~-~t~ri~ at the boKom of the
flask) in PBS for 10 min at 37~C in a tissue culture flask. The cell monolayer was
coll~cterl, washed with PBS and deposited onto microscopic slides using a
s Cytospin centrifuge at 800 x g for 5 min. Cells deposited on the slides were fixed
with 4% ~a.~." "~ hyde for 15 min at room temperature and then stored in 70%
ethanol at 4~C until use.
B. In situ hybridization on slides. Slides stored in 70% ethanol prepared as
o described hereinabove were rehydrated in PBS for 10 min. The slides then were
treated with 1 ,ug/rnl proteinase K in 20 mM TrisCl + 2rn~ CaCl2 buffer for 15min
at 37~C, followed by two washes with PBS. The slides were fixed again with 1%
paraformaldehyde for 10 min followed by two washes with PBS. Five (5) pmol of
probe 7 (SEQUENCE I.D. NO. 7) dissolved in PBS + 0.1% SDS was applied to
15 each slide and covered with coverslips. Hybridization was carried out in a
hnmic~ifierl box at 37~C for two hours. After hybridization, the slides were washed
twice with PBS pre-wa-rmed in 45~C water bath. The slides then were stained with2.5 ~g/ml Hoechst 33342 in PBS (Sigma, St. Louis, MO) for 5 min and washed
once with PBS for 5 min. The slides then were mounted with VectaShield (Vector
Laboratories, Bllrlin~rn~, CA) and viewed under microscope.
C In vivo infection. Groups of CF-1 (outbred) mice (obtained from Charles River
Laboratory, Wilmington, MA) were inoculated intraperitoneally with 1 x 106 E.
coli.. Blood from mice (approximately 1 rnl)was collected by cardiac puncture at2s various times (5 min, 15 min, l hour, 2 hours, 3 hours, 6 hours, 8 hours and 12
hours) after inoculation. The blood was separated using 6% dextran gradient,
washed and fixed as described in the above text. In situ hyhri-li7~ti~-n was
performed using Probe 7 (SEQUENCE I.D. NO. 7) as described above. PMNs
isolated from infected mice were hybridized with fluorescein-labeled PNA probes
targeting bacterial 16S rRNA. Fluorescein signal was collected with FITC visual
filter cube for Nikon microscope (Fryer Co.Inc., Huntley, IL) with 480/30 nm
exiciter filter, 505 DCLP dichroic mirror and 535/40 nm eInitter filter.
D. Results. In both in vitro and in vivo systems, we have demonstrated that PNA-3s FISH technique described in this work can be used for diagnosis of bacteremia or
sepsis. FIGURE 3 clearly shows that bacteria E. coli was brightly stained with
FrrC-labeled PNA probe (SEQVENCE I.D. NO. 7) six hours after the rnice had
CA 02221179 1997-11-14
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29
been inoculated with bacteria E. coli. Also, at the inoculum level of 1 x 106 E. coli
per mouse, bacteria in the bloodstream were detP-ct~Pclwith the PNA-FISH technique
five minlltPs through 12 hours after inoculation, when the mice started to die. The
PNA-FISH assay thus can be used for the iclentific~tion of gram positive, gram
negative bacterial or fungal infections, in a very short turn-around time (4-5 hours).
This technique thus is very sensitive and provides valuble information to the
physicians for the djagnosis of becterernia and sepsis.
Example 10. Morpholino Probes and their use in Fluorescent In Situ Hybridizationlo and Solid Phase Capture
A. In Situ Hybridization. As described in Example 1, morpholino oligos can be
used as probes for fluorescence in situ hybridization. A morpholino oligo
(SEQUENDE I.D. NO. 4) is labeled with fluorescein at the amino-end of the
15 peptide and is used to detect the complimentary sequence of 28S rRNA following
the FISH assay as disclosed hereinabove in Fx~mple 1. The optimal morpholino
hybridization conditions are determined as described in Examples 1, 2, 3, 4 and 5.
EPICS(~)Profile II (Coulter Corp., ~i~ h, Florida) flow cytometer, equipped withan Argon laser, is used to detect the probe. The laser is set to 15mW at 488nm,
20 with fluorescein fluorescence acquired using the light selection filter of 525/30 nm
(central wavelength/full bandwidth at half-maximal tr:~ncmiccion).
B. Solid Phase Capture. Morpholino oligos (e.g., SEQUENCE I.D. NO. 4) can be
affixed to a glass surface and complementary nucleic acid (DNA or RNA) detectP-l25 as taught in U.S. patent application Serial No. 08/311462, previously incorporated
herein by reference.
Bacteremia is a serious and urgent disease caused by the emergence and
habitancy of various bacteria in blood. An individual may die within several hours
30 to several days. Clinicians need rapid test results to confirm blood infection, to
identify the invading organism and to administer an effective antibiotic to the
individual. Currently, blood infections are tested using blood culture bottle method
followed by grarn stain. The current procedures take a day to a week, and
somPtimPs even longer, for org~nicmc that are hard to culture. Fur~hPrm~ re, false
35 negative culture data can result in individuals who receive antibiotic therapy prior to
or during sample collection.
CA 02221179 1997-11-14
W 096/36734 PCTrUS96/07075
Using the PNA-FISH technique we developed, we can detect bacteria in -
phagocytic cells in peripheral blood. Polymorphonuclear nt;uL.uL.llils (PMNs) form
our first line of defense against invading bacteria. They serve as conc~ tldlol~ for
bacteria. Fluorescently labeled peptide nucleic acid probes have been made
s targeting b~teri:~l 16S rRNA. Using an in situ hybridization technique and PNAs
(or morpholinos), bacteria inside PMNs can be brightly stained. This technique
thus provides a rapid (4 hour) assay for detection of 1~cter ~l in test sz-mpl~s
-
CA 0222ll79 l997-ll-l4
WO 96/36734 31 PCTrUS96/07075
~Qu~ LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: YU, HONG
DUNN, DAVID A.
VALDIVIA, LYNN
(ii) TITLE OF lNV~N'l'lON: POLYMERIC PEPTIDE PROBES AND USES
THEREOF
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(A) TELEPHONE: 708-937-6365
(B) TELEFAX: 708-938-2623
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) sTR~Nn~nN~s: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
- (A) NAME/KEY: 5' ~luorescein
(B) LOCATION: 1
(ix) FEATURE:
(A) NAME/KEY: 3' ~luorescein
(B) LOCATION: 25
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
ACCTTTTCTG GGGTCTGATG AGCGT 25
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W 096/36734 32 PCT~US96/07075
(2) INFORMATION FOR SEQ ID NO:2:
(i) ~QU~N~ CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: dou~le
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: 5' fluorescein
(B) LOCATION: 1
(ix) FEATURE:
(A) NAME/REY: 3' fluorçscein
(B) LOCATION: 25
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
TGACAGCTTA TCATCGATAA GCTTT 25
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
~ (A) NAME/REY: 5' fluorescein
(B) LOCATION: 1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
GCTGCCTCCC GTAGGAGT 18
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs.
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/REY: 5' fluorescein
(B) LOCATION: 1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
AC~ G GGGTC 15
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
~ CA 0222ll79 l997-ll-l4
W 096/36734 33 PCTrUS96/07075
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: 5' fluorescein
(B) LOCATION: 1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
CTGGGGTCTT TTCCA 15
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: 5' ~luorescein
(B) LOCATION: 1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
GCTGCCTCCC GTAGG 15
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: 5' ~luorescein
(B) LOCATION: 1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
GCTGCCTCCC GTAGG 15
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) sTR~Nn~nN~ss: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: 5' ~luorescein
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W 096/36734 34 PCTrUS96/07075
(B) LOCATION: l
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
GGGCATCACA GACCT l5
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STR}~Nl )~nN~c:S: double
(D) TOPOhOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: 5' fluorescein
(B) LOCATION: l
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
GTGGAGTAAC CTTTT l5
