CA2049043A1 - Immobilized oligo-nucleotide probes and uses therefor - Google Patents

Abstract

The invention is a method of detecting nucleic acids in a sample using oligonucleotide probes which are noncovalently bound to solid supports for rapid, sensitive, hybridization assays. The method involves coating the support surface with a polynucleotide and then hybridizing a specific capture probe for each analyte to the polynucleotide by way of a short tail of the complementary polyunucleotide. The immobilized probes are used to capture nucleic acid targets out of complex specimens for nonisotopic detection without the need for prior cell culture or purification of the target nucleic acids. A panel of tests can be run on each specimen simultaneously, a format that conserves precious samples. The assay can be readily automated, and can be conveniently run in a manual fashion on large numbers of samples in two to three hours.

Description

W090/10716 2 ~ 3 PCT/US9OtO1205 IMMOBILIZED OLIGONUCLEOTIDE PROBES
_______________________.___________ AND USES THEREFOR
_________________ :

Back~round Biological samples generally contain nucleic 05 acid sequences which encode information unique to - its biological source. For example, all species of bac~eria which belong to a certain genus (e.g., Cam~lobacter or Enterobacter) share certain physical characteristics which are encoded by the 10 same nucleotide sequences present in most or all of the species within the genus. Thus, an assay which is specific for the ~enus can be based upon these common nucleotide sequences.
Detection of nucleotide sequences in a sample 15 can be carried out using nucleotide probcs speclEic ~or these targot scquenccs. For examplc, Na~ a ~t al., FEBS, 183:379-382 (1985), doscribe tho uso oE
UV irradiation to bind h~torolo~ou~ hi~h molnculnr weight DNA to polystyrene microtiter wells in order 20 to detect speaiflc sequences within the immobilized DNA by way of specific DNA probes.
Zouali and Stollar, J. Immuno. Methods, 90:105-110 (1986), describe a technique for the attachment of high ~olecular weight nucleic acids to 25 polystyrene microtiter wells usin~ pre-treatment of the support with UV irradiation.
Polsky-Cynkin et al., Cl~nical Chemistry, 31:1438-1443 ~1985), describe the use of immobilized ~ capture probes in clinical assays.
Kremsky et al., Nucleic Acids Research, 15:2891 2909 (1987) and Wolf et al.,.Nucleic Acids Research, 15:2911-2926 (1987) describe a technique :, ________ __ 2~ q~ 2-for the covalent attach,~nent of oligonucleoeides to latex coated polystyr~ne beads.
Stabinsky, U.S. Patent Number 4,751,177, descrlbes a single-step target capture that utilizes 05 B hybridizatiDn of a tailed capture probe in solution followed by ~i solid phase capture with oligo-(dT)-controlled pore glass.
Soderlund, UK Patont Application GB 2169403A
(1985), describes several affinity-based capture hybridization methods which use two probes, detector probe and a capture probe that cQntains one rnember of an affinity pair.
Collins, European Patent Application Number 265 244, describes a noni~otopic reversible target capture protocol which makes use of dA-tailed oligonucleotide probe~ and ol~go~dT)-magne~ic particles and poly~dT) Eilt~rs.
Presently avallabl~ nonisotopic as~ny m~thod~
are either lacklng in sensitivity for certain applications, or are too complex or too slow to be clinically use~ul. They also r~quire a sample to be split in order to perform multiple assays thereon, resulting in decreased sensitivity. Most of the prior art methods also employ solid phases that are not easily separated fro~ ~iscous clinical samples such ais stool. It would be helpful to have a rapid, nonisotopic assay useful for assaying complex or u~ipu~ified-samples that is highly specific, simple .
to use useful ~ith RNA as-well`as DNA targets and applicable to olinical and food samples with no prior purification of the nuclèic acids.of the samples. '~

::,- ' '' ' .:. . . .` ' ' . '.:: :'., ' ' '': : ' .' : : `.',` : ' ' ' W090~l0716 PCT/US90/012D5 -3- 2~ 0@~3 Summary of th~ Invencion ______ _~_______________ The present invention pertains to a method of determining (detecting and/or quantitating) target nucleic acid sequences in a sample, which is simple 05 and rapid and does not require the use of radio-active materi~ls. In the meth,od of the present invention, oligonucleotide probes ~capture probes), which are speciflc or nucleic ac~ds to be detected (target nucleic acids), and bound to an appropriate 10 support, are contacted with a sample to be analy~ed for the target nucleic acids, under conditions appropriate for hybridization of complementary nucleic acid sequences to occur. In general, the sample has been previously treated in such a manner 15 that the molecular structur~ of th~ c~ s ls disrupted (i.e., the c~ll istructure, i~uch ns thc chromosom~s and m~branois ~ro broken, and tho cellul~r cytopl~m ii3 ~ispcri~od).
In the method of the pre~ent invention, a 20 sample is treated to r~lease the nucleic acids of cells contained in the sample, nnd is combined with a capture probe, which is reversibly attach~d or preimmobilized on a support, such as polystyrene, by means of a ho~opolymer tail whose sequence is 25 complementary to a sequence present on the support surfaea. Hybridi~ation of complementary nucleic aeid sequences results in capture of target nucleie acids from the sample. Capture of the target on the ?
solid supports also sierves to separate the target 30 nucleie aeids from sample impurities prior to nonradioisotopic or radioisotopie detection. Target W090~07l6 PCT/US90tO1205 3 ~ ~

nucleic acids can be de~ected and~or quantified by hybridizing the captured target with a labeled probe, for example.
The present method has numerous advantages over 05 presently-available ~ethods. For example, the present method makes it possible to: (l) analyze m~ny samples (e.g., 20 or more) nonisotopically in a short time; (2) carry out ~nAly3es without sample filtration or cell cultur~ or prior purification of 10 nucleic acids; (3) run a panel of tests on a sample simultaneously without crosstalk; (4) run multiple tests on a small volume sa~ple; and (5) use capture probes without prior puriEication. In addition, the method can be efficiently pcrformed using a single 15 labeled probe, since one generic probe can b~
constructed (~.g., by clonlng) to hybrldizo to all target nucleic ~cids tha~ m~ke up n screen or n panel. Tho pre~en~ ~sa~y ma~hod ~llows tho non-radioactive detectLon of as little ns one 20 hundred attomoles of target nucleic acid in any type of cell extract (bacterial, mammalian, yeast, plan~), in food and clinical samples and other impure biological specimens in about two hours.
In addition to its use in de~ecting and/or 25 quantitating the level of target present in a sample, the present invention can also be used, without the customary phenol extraction, to isola~e nucleic acid targets from crude specimens for cloning (or subcloning) and~or amplification.
30 Substantial purification of the target prior to either cloning or~ target amplification (such as the :: ` :` ' . '' :. : ' ' . .`; ` .:.',' ' .'.. ~ : , ': `

W090tlO716 PCT/US90/01205 2 ~ L~

PCR method, Mullis, U.S. Patent 4,683,202) reduces the level of background (and thus improves the ~.
specificity) of these procedures as well as removing numerous interfering substances present in crude 05 specimens. In addition, if necessary, the sensitivity of the detection of targets by the present mathod can be substantially improved by insertin~ an optional target amplification method between target capture and detection or by using ~.
reverslble target capture methods as disclosed herein.
The method of the present invention is particularly useful for precious or small volume samples because it i9 not necessary to divide the sample into smaller samples for each tost to bc carr~ed out, Anoth~r adv~nt~ge o~ the pr~ nt in~ention i9 th~t A non~pooiic or gcnaric roportor probe c~n be u~od boc~u~e tho po~iblo lo~ of signal due to the presence o~ interfering substances 20 or high levels of competitor organisms is avoided by capturing target nucleic acid sequences from the sample being tested prior to labeling.
The present in~ention also includes kits for rapid analysis of samples by the method of the 25 present invention. A kit can contain, for example, suitable solid supports, such as dipsticks or wells, which contain a substratum,.which is discussed in greater det~il hereinbelow,: and a specific capture - -probe prehybridized to the substratum, and an agent, ~:
30 such as a.lysis solution,.for disrupting cells to ~-free cellular nucleic acids for:detection.

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W090/10716 PCT/US90/0120~
2 ~ L~ ~ O d~

Alternatively, the kit can contain capture probes ., which are not bound to the solid phase. This requires the user to perform the prehybridiza~ion step. Such a system allows the user more 05. flexibility since the user would prepare the appropriate capture probe-solid phase adducts whenever dasired. The kit can, optionally, contain a labeled probe and n means for deteeting ~he labeled probes, positive and/or negative control 10 samples, elution buffers for carrying out reversible target capture and amplification or cloning reagents.

Brief_De9C_i~tio__o_ _e~FigureS
Pigure 1 depicts the rate of eapture oP rRNA
targets with ~A) the capture probe ~ree in solutlon;
15 or (B) wLth the captura probe prehybridizo~ to tho polystyrene.
Figure 2 ~epict3 tho ~o~o-respon~o eurvo for the nonisotopic deteetion of Salmonella ty~himurium in 25~ w/v normal stool with the present invention.
Figure 3 is a schematic representation of the nucleotide sequence of the oligonueleotide probcs used in the present invention.

Detailed Deseri~tion of the~ I_vention The present invention relates to a rapid, 25 nonisotopic method of determining (detecting and/or quantitating) nucleic acid.sequences- of interest (target nucleic acid sequences) in a sample, as well as a method of isolating..or purifying target nueleic acid sequences (e.g., for the~.purpose of i f3 ~ 3 ampllfication o~ cloning), materials useful in carrying out the method and kits containing such materials, useful for deter~ining target nucleic acid sequences in a sample.
05 In the ~ethod of the present invention, a complex sample to be analyzed for the presience of one or more of the target nucleic acid sequences is combined with A solid ~upport which has affixed to or im~obiliæed on its surace nucleic acid sequences, or probes, referred to as "capture nucleic acid sequences" or "capture probes". The capture probes are complementary to the target nucleic acid sequences, and, under appropriate conditions, will hybridlze with the target nucleic lS acid sequences, The c~pture probes ~re bcund to a poly-nucleotide lay~r or ~ubstratum, which is discui~s~d in greater detAil below, which i9 contc~ ~iractly onto the solid support, through a "tail" which is 20 complementary to the substratum.
The preisent method can be used on any sample which contains nucleic acids. The sample must be sufficiently liquid to allow contact with the probe and for hybridization to occur. The sample can be 25 complex, clinical samples, such as, mucus, sputum, urine, stool, or blood, or foods or beverage, such as milk, cheese, wine or prepared foods. The sample does not ha~e to be separated, filtered or precultured prior to use of the present method. The 30 sample is generally pretreated with an agent which disrupts molecul-ar structures withi'n the cells.

WO90tlO716 PCT/US90/01205 2 ~ L~

These agents are referred ~o as '~agents that disrupt molecular structures" (e.g., "chaotropic agents"), and will disrupt the cells, or viruses, present in the sample to release nucleic acids. Such agents 05 are generally compounds or solvents which disrupt the molecular structure of a cell, that is, these agents are capable of denaturing the secondary, tertiary and/or quarternary structures oi blopolymers, including proteins, nucleic acids and 10 polysaccharides, that are generally found in specimens. Examples of agents that disrupt molecular structures are chaotropic salts (e.g., guanidinium thiocyanate), and monovalent salts of large acidic anions (e.g. trichloroacetate, 15 trifluroacetate), dennturing detergents (e.g., dodecyl sulfate), hydrolytic ~nzymei~ (o.g., proteases), nnd compounds which disrupt hydrophoblc bonds (e.g., phenols, dLmcthyl formAmid~, dimethylsul~oxido, tetramethyl -r~n, ~unnidlnlum 20 hydrochloride) or hydrogon bonds (e.g., urea, formamide). Physical or mechanical means of disrupting molecular structures, e.g., sonication, can also be used to release nucleic acids. Agents that disrupt molecular structures can be used singly 2$ or in various combinations to achieve a desired result.
Solid supports which can be used in the present invention include any solid material- to which can be bound sufficient amounts of the substratum poly-30 nucleotide to allow the capture probe to be pre-hybridized to the support. Polymeric materials, such as agarose beads or polystyrene, are generally useful as supports.

Polystyrene is a particularly useful support for use in the presen~ invention because ie can bind large amounts of polynucleotide substratu~ material, allows facile nonisotopic detection of targets, and 05 has relatively low nonspecific binding of both enzymes and nucleic acids By "nonsp~cific binding"
is meant the target~independ~nt binding o a probe to a solid support. The forces respon3ible for the sticking are not well understood (h~nce the use of the word 'nonspecific'), but van der Waals bonds, hydrophobic bonds, and hydrogen bonds are thought to be likely contributors to the total energy of the binding. The labeled probe may b~ uncomplexed but more likely is complexed with one or more cellul~r components that enhance the tendoncy of the probo to bind to the solid support. It is measured by incubating thc sampl~ or ~p~cimen, tho label~d probe, the cnp~ure probc, and ~olid support in the absence of the target, and in the absence of any "pseudo targets" ~i.e., targets closely resembling the actual target) with which nonspecific hybrids may form. The configuration of the support will vary depending upon the type of assay`and the nsture of the samples to be assayed. Confi~urations such .
25 as microtiter wells, tubes and dipsticks are useful :~
in the present invention and allow the simultaneous assay of a lar~e number of samples to be performed -manually in an efficient and convenient way. The assay can also be automated using, for example, microtiter.wells.and is.capable of extensive automation because of automatic pipetters and plate .

.

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i 9 ~

readers. Other solid phases, particularly other plastic solid supports, may also meee these basic criteria (including the abllity to adsorb large amounts of polynucleotide) and thus be suitable for 05 use in the present invention.
A substratum or layer is coated onto the supports. The 'substratum' is a layer of material that is laid down on a solid support by covalent or noncovalent m~ans, which greatly enhances thc 10 binding of the next layer of ma~erial. The substratum can be any polynucleotide. The polynucleotides can be from about SO to about 3000, or more, nucleotides in length. About 1500 to 3000 nucleotide bases is a particularly useful len~th.
15 Preferred substrata materinls are homopoly-nucleotides such as homopolymcrs of deoxyribothymldylnto ~dT), daoxyriboadenyltlto (clA), d~oxyribocyfidyl~te ~dC~ or deoxyrlbo~unnyln~o tdG).
Poly(dT) and poly(dA) ar~ p~rticul~rly preParrad 20 substrata for the pr~sent invention. However, mixed sequences comprlsing simple repeating polynucleotlde sequences, having repeat sequences no longer than about a tetranucleotide, can be used. For example, alteratin~ nucleotides (e. e . GTGT or GGTTGGTT) can 25 be used as the substratum, wherein the length of each repeat sequence is four nucleotides or less.
In ~eneral, about 250 n~ to about 1 ~g of substratum is bound to the support.
Bindin~ a substratum or layer of material to a 30 solid support (such as polystyrene) allows the --binding of a mu~tiplicaty of oligonucleotideicapture : :. , ,:: : - : ~

probes (up to about l ~g), which in the absence of the substratum do not bind to the solid support (< l ng to polystyrene). More than one substratum layer can be bound to a support. For example, large 05 adaptor molesiules containing short tails, as well as a multiplicity of properly spaced secondary ligands, csn be bound to a poly(dT) (or other polynucleotide) substratum bound to polystyrene. The ~ultiplicity of secondary ligands constitute the second layer or lO second substratum. Thus many first probes or other adaptor molecules can bind to the solid support and ~-each of them would be capable of binding a plurality of capture probes. Additional layers can be added as needed. In thls way the cnpacity of the solid lS support can bs increased to any des~red levol to improve the speed ~nd ~fflclency o~ tho cnpture o~
target ~ol~culcs.
The cS~pture probe~ aro mixod b~sQ nucloic ~clcl sequences, which ~rc specific i~or the target nucleic 20 acids to be detected. The mixed base sequences of the probe allows it to base-pair only with the complementary sequence of the target nucleic acid, under appropriate conditions o~ stringency. That is, the capture probe may bind "pseudo targets"
25 which have sequences which are closely, but not exactly complementary to-the mixed base sequence of the probe, forming nonspecific hybrids. Nonspecific .
hybridization can be reduced under stringency `-conditions. (Stringency is described below, and in 30 detail in Example 6). The specific sequence of the capture probe will depend upon, and be complementary - . . . ~ s WV90~1~7]6 PCT/US90/~12~5 2 ~
-l2-to, the nucleic acid sequence of the target nucleic acid. The probe can be DNA or RNA.
The capture probes are bound, via a ~'tail", to the subseratum present on a solid support. The tail 05 is an RNA or DNA sequence that is complementary to the substratum. The tail can be added either synthetically or enzymatically to either the 5' or 3' end, or middle, of the capture probe. Thus, if the substratum is poly(dT), a complementary tail 10 would consist of poly(dA). Oligonucleotides (i.e,, about 10-50 nucleotides) are preferred as capture probes over polynucleotides (i.e., more than about 50 nucleotides) because of th~ ormer's greater specificity. Oligonucleotides can readily dis-15 tinguish a p~rfectly formed double h~lix (oxactmatch) from ~ust a 5ingla-base pair mismatch. Thl9 exc~ptionsl sp~ciElci~y ha3 bocn tormad "nllal~
speciicity". Connor, 8.J, ot al, (1983), Proc.
Natl. Acad. Sci. USA, 80, 278-282 describe the 20 allele-specificity of oligonucleotides generally.
Collins snd Hunsaker, (1985), Anal. Biochem., 151, 211-224 describe the allele-sp~cificity of tailed oli~onucleotides.
The tailed capture probe is reversibly or 25 noncovalently bound to the substratum. That is, the binding of the tail to the substratum can be changed by a simple physîcal stress, such as increasing the te~perature or the chaotrope concentration. Thus, capture probes bound to a poly(dT) substratum via a 30 (dA~ tail can be readily remo~ed, after binding with the target nucleic acid for further processing.

.

2 ~

This can be done for example, simply by adding heated buffer to the polystyrene or increasing the concentration of a chaotrope or nucleic acid de-naturant above 8 certain threshold level. The oS probe-target complex can be recaptured by cooling, or by a simple dilution of the nucleic acid denaturant.
In one embodiment of the present invention, UV
or gamma rndi~tlon i9 used to bind the homo-lO polynucleotide substratum, such as poly(dT), tosolid supports, such as microtiter wells, dipsticks, ~nd tubes. The process of binding a polynucleotide substratum to a substrate is described in detail in Example l.
The ~iled capture probe is im~obilized on the substratum by prehybrid~zing the probe with tho substretum. That is, th~ t~ilad probe ls hybrLdL2~d to tho substr~tum complo~nt~ry ~oquenco prior to use in ~n ~ss~y. In the prefarred method, the 20 substratum sequence ls poly~dT) and the tail is poly(dA). However, other simple repeating sequences can be substituted for the dA-dT afinity pair. The repeating sequences should preferably be no longer than about a tetranucleotide (i.e., 4 nucleotide 25 bases) in order to provide the rapid hybridization kinetics that would be~desirable in a manufacturing -~
process. In prior-art methods, the tailed probe ls added by the user to tha specimen, and bound to the ~ target in solution and then bound to the solid 30 support. That format is-disadv:antageous in~that multiple tests canno~ be performed`on each sample, I

W090/l0716 PCT/US90/01205 2~ O~
- 1~

and the tail on the capture probe must be long to overcome steric hindrance in the approach of the target to ths support. In the present method, the user simply combines one or more solid supports, 05 already containing bound capture probes, and the sample and maintains the resultin~ combination under conditions appropriate for hybridization of coMplementary sequences to occur.
More than one type of immobllized oligo-10 nucleotide capture probe can be contacted with a single sample at one time, thus making it possible to analyze the sample for more than one target nucleic acid sequence nt a time. For example, different solid supports qpecific for different lS bacterlal genus~s can be u~ed ~imultaneou~ly to t~clt one sample for the prosence of thc bactcria. Tho stability o~ thc linkn~e botweQn tho prob~o nnd th~
substratum substantially pro~ont3 croqs~hybridization, or "cross-talk", among the 20 diiferent type~ of probes. The term "crosstalk"
means interprobe exahange of capture and or target oligonucleotites. That is, where one or more dipstick probes is contacted with a sample, a capture oligon~cleotide bound to one of the dip- .
25 sticks, which may or may not be hybridized with a target nucleic acid, detaches.from.the dipstick and becomes bound to another dipstick in the-sample.
- This migration of probes between dipsticks is -"crosstalk". The term ca~.japply-to other support 30 configurations besides-dipsticks,..which was used merely to illustrate the present.exampLe.:. ... , W~90/10716 PCT/~S90/01205 Prehybridizing the capeure probe to the support eliminaees the need to purify the capture probes after the tailing reaction. Only oligonucleotides with a tail of sufficient length will bind to the 05 coated polystyrene.
Prehybridization also helps to overcome potential interference from endogenous poly(A) or poly~dA), which Lnt~rference ~s potentially signiicant in clinical samples containing a large number of human cells. Capture probes with very short tails can be used since the target cannot sterically hinder the tail's attempt to bind to the solid support. The tail length need only be sufficiently long to form stable hybrids with poly(dT) in the buffer employed. Reduced ~ail length also dacreas~s th~ co~t of the c~pturc probes. The pr~E~rred tail lon~th ls Prom ~bout 15 to about 30 nucleotides.
In the m~thod of the presont invention, `-20 oligonucleotide capture probes preimmobilized on ;
solid supports by way of the homonucleotide affinity pair are contacted with the sample for direct detection of specific microorganisms in the samples.
For example, Cam~ylobacter has been detected directly in clinical stool samples with about 9O~
specificity and sensitivity. The assay can detect Cam~ylobacter and Salmonella seeded in 25% wJv normal stool at less than l.O x lO5 cells/ml.
~ The method described herein simplifies sample 30 handling and processing in that filtration normally required for stool and other ~iscous or chunky : :'. ~ . , WO~0~10716 PCTtUS90/01205 2 ~ ~9 ~ '1 3 - 16-samples is omitted and pipetting can be minimized due to the ability to add one dipstick for each test to every sample. The prei~mobilization of the capturP probe on a solid support allows simultaneous 05 analysis of each and every clinical sample for a number of differen~ analytes.
In the present method, specimens are first dis-solved by a chaotropic agent ~e.g., a sol~ent disrupting molecular structures). By doing capture lO in a chaotropic solvent which disrupts the molecular structures of the target cells or viruses prior to labeling, other substances that could potentially interfere with this nonisotopic assay, such ns endogenous biotin, avidin, isvidin-llke moloculea, 15 nucl~ases, protoas~s, and nucl~ic acid binding proteins, ar~ removed prlor to tho introduction of the label~d g~ncric probe. ~ numbcr oE immobillxo~
capture probes, for oxn~plo, polystyrone dipsticks, each with a diffcrent specific capture probe 20 (includin~ an optional nonsense control sequence), i9 then placed into each sample to remove tar~et molecules complementary to each of the probcs. The dipsticks are removed from the specimens, washed, labeled, for example, with a generic reporter probe, 25 and detected nonisotopically. Nonisotopic detection can be carried out using the biotin-streptavidin-alkaline phosphatase system described by Leary et al. in Proc. Nat'l. Acad. Sci. USA, 80:4045-4049 (l983). The tar~ets can also be labeled with labels 30 such as fluorophores, che~iluminescent compounds or radioisotopes and detected using standard methods. `

.: ~ . .. . . ... . ..... . ......

The present method optionally includes a "stringency" wash step. A stringency wash is a ~ash protocol thac reduces nonspecific hybridization. A
n~nspecific hybrid is an i~perfect hybrid, that is, 05 one that contains mostly correct base pairs, but has one or more mismztched bases (e.g., A not paired with T or U; C not paired with G). Nonspecific hybrids generally have a lower dissociation temperature than the per~actly base-paired hybrid 10 between the actual target and the capture probe.
Nonspecific hybrldization results in a complex of a capture probe and a "pseudo-target", which is a molecule that closely resemblas the actual or real target in its base sequence. The temperature of a 15 stringency waish is generally about 1-5~C below the dis~ociation temperature of the capture probe-tar~t complex, but ~ust above th~ dlssociation tompora~ur~
o~ any capture probe pseudo tar~at complcx~i~ th~t may be present. A quatcrn~ry salt, such ~g 20 tetraethyl ammonium chloride (TEA Cl) can be used ~or the stringency wash. For example, at a concentration oP 2.4M, TEA Cl confers equi~alence to G-C and A-T base pairing, allowing the stringency oP
hybridization to be controlled at a single 25 temperature (for a fixed probe length) in a manner that is completely independent of GC:content. Wood, .I., et al., (1985), Proc. Natl._Aicad ~Sci, -: -82:1585-1588. Since TEA Cl strenghtens A-T base pairing in relation to G-C, lt was employed as a 30 waish reagent to maintain tight binding-of:the dA-~ailed probr eo~the poly (dT) coated support .. ~ . '~

,' wo 90/10716 Pcr/us90/01205 3 ~ ~ d' - 1 8 -while decre~sing the strength of mismatched hybrid-ization reactions between the mixed base sequence oligonucleotide capture probe and any nucleic acid sequences closely resembling the true target. Two 05 surprisinK sspects of the TEA Cl wash in the present system sre that (1) non-specific binding of the labeled probe to the aolid support is dècreased while, ~2) hybridization si~nals are increased when the wash i9 applied prior to the addition of labeled 10 probe.
It has now been shown that the level of subsequent binding of the labeled probe to captured targets is increased by up to a factor of eight when the TEA Cl wash step ls applied prior to the ad~
15 dition oE the labeled probe. Washing with TEA Cl prior to the ~ddition of lnbeled probe c~us~ n decrease in the level oP tho nonspeciPLc bindlng oE
the labeled probe to the support du~ing the sub-sequent labeling reaction. Similiar results ha~e 20 been demonstrated with 2.4M TEA Br and can be predicted with the use of related salts/solvents.
Without wishing to be bo~nd by theory, it i9 believed that the TEA cations remain bound to the poly(dA)-poly(dT) for a significant period of time 25 and somehow alter the structure of the immobilized probe-target complex with the result that the target becomes more accessible to incoming riboprobes, thus increasing the hybridization efficiency. For example, the target^rRNA may have its:elaborate 30 secondary structure-relaxed in TEA -Cl.~ Orosz and Wetmur, (1977), Bio~olymers, 16:1183 1199 describe 2 ~
- 19- `

the ~elative instability of the RNA-RNA double hel~x in TEA Cl. For a short period of time the target may be more accessible to the incoming riboprobe, causing an overall increase in the level of 05 hybridization.
In additlon to the format described above, the present ~ethod can be used in a "revers~ble target "
capture" format. By "re~ersible targe~ capture" is meant the following process: A target ig lO immobilized on a first solid phase. The flrst solid phase is washed and the target is ramoved by addition of the first elution buffer and immobilized on a second solid phasè. The second solid phase is washed, and, optionally, the target is remo~ed with lS a second elution buffer and immobilized on a third solid pha~e. The proce~s can be repe~ted ns often as desired. The proces~ oE reversible tnrget capture greatly onh~nce~ ~ha signnl/noi~e ratio oE
the hybridix~tlon ns~y. Tho ~olid phnisos can be 20 the same or different; the elution buffers can be the same or different. Generally, maximum signsl/noise ratio~ are achieved when both the solid phases and the elution buffers 2re different.
Formats for re~ersible capture include, for 25 example , ~
l. Targets are captured with short-tailed (e.g., ~ ~-having a dA tail about lS nucleotides long) oligomer - probes (about 35-50 nucleotides in length) which are prebound to.polystyrene, in a tetraaIkylammonium 30 salt. If desired, a specific labeled probe can be W~90/10~16 PCT~US90/0170S
2 ~ 3 added during the first capture. If a generic labeled probe is desired, it can be added to the elution buffer. Elution is performed using 2.5 M
GuSCN and recapture is done with a second 05 oligon~1cleotide containing a longer tail (e.g. ~
dA-50 or dA-lO0 tail) which will form stable hybrids with poly~dT) in 2.5 M GuSCN. (2 oligoprobe specificity, 3 i a specific l~beled probe was used).

lO 2. Targets are captured with short-tailed (e.g., about 15 nucleotides) oligomer probes te.g., about 35-50 nucleotides in length) prebound to polystyrene in a tetr~alkylnmmonium 3alt. If desired, a specific labeled probe can be ~dded during th~ first ' 15 capture. If ~i goneric li~boled probe i9 doslrod, it can be addod to the olution buffor. Elution i9 performed with 2.5M GuSCN. GuSCN i9 then diluted so that the short tailed probe can rebind to poly(dT).
(l oligo probe speclficity or 2 if a specific labeled probe was used).

3. Targets are captured with a short oligomer (e.~.
about 15-20 nucleotides) and a long tail (e.g., dA-lO0 or more). A specific labeled probe can be ; ' used during the first capture; a generic labeled 25 probe is best used during the elution. The tar~et is then released by,contacting with a tetraalykylamm~nium salt that stabiliz'es poly(dA)--poly(dT) ,relative to mixed base sequences.
The tailed probe remains bound to the suppor~; the '' ' ' ,'~

2~

tar~e~ is releas~d. The target is then recaptured with a se~ond specific oligomer eo i~prove the specificity of the hybridization and reduc~ th~
nonspecific bindin~ of excsss labeled probe to the ~ ~ `
05 solid supports. (2 oligo probe specificity, 3 if a specific labeled probe was used).

4, In the preferrod method, the first capture probe is prebound to the flrst solld support. A second ~ixed base sequence can be prebound to the second lO solid support, The next step is to capture the target on the first solid support. A specific labeled probe is beqt added during the first cnp~ure while a generic labeled probe is best addad to the elution buffer. Elution is dona in such a way a~ to lS remove only the target, or the tnrget and the fir~t capture probe. It i8 preEernbl~ to ndd ~ speclflc labeled probe to the olution buPfcr lf ~he process of elution would dissociate a previously added labeled probe fro~ the target. The sample is either 20 cooled or diluted, as required, and added to the second solid support. The poly(dT) of the second solid support was pre-saturated w~th the second capture probe well enough to prevent any significant rebinding of the target to the second second support 25 through the tail of the first capture probe. In -other words, the target is forced to rebind to the support by hybridization to-~he second specific I ~;
~ixed base sequence (2 oligo probe specificity,or 3 oligoprobe specificity if a specific oligo~er ~ 30 labeled probe was- used). ` ~

. :, . . - ' ~ .: ;;.`, .

:, ' ' : ' `', ~, : '.' ", " '" . ' , ', ' .', , " ,, ' : ' ` ` ' "

WO g()/10716 PCl'/US90/0120s 2'~ 22 By adding an appropriate amount of poly(dT) to the elution buffer, the poly(dA) of the first capture probe can be prebound to the poly(dT) in solution before it has a chance to bind to the 05 second support. This method has been employed successfully to block poly(dA)-containing molecules from binding to poly~dT) supports containing prebound tailed probes in a co~pending U. S. patent application entitled "Methods and Apparatus ~seful 10 in Preventing Endogenous Substances from Interfering with Affinity Capture Schemes" by Collins, Gillespie and Morrissey, Serial No. 07/322,419, fil~d March 10, 1989, the teachings of which are incorporated herein by reference.
Poly(dA) can be incubAted with tho s~cond ~oli~l phase prior to ~he nddition of tho s~mplo thAt eluted irom cho ~irst solit ph~ with tho Eir~t tailed probe. This will cffectively preblock any free poly(dT) on the second solid phase so that no 20 poly(dA) from the fir5t tailed probes can bind to the second solid support.

It should be noted that the procedures de-scribed above are not limited to just two captures, -~
but can be extended to any desired number of 25 captures. However, if more than two are employed, the assay is more suitable to automation than for manual use. In addition, the second or even the third solid phases (with triple capture) do not have to be of the same chemical composition as the first.
30 Indeed, advantages have been disclosed for the`use of very different solid phases in reversible target capture (Collins, European Patent Applic~tion Number 87309308.2).

WO90/1071b PCT/US90/01205 . The assay steps of the present method can be ; readily automated. This Ls especially so with microtiter plates as the solid phase, since automatic pipeting equipment (for reagent addition 05 and washing steps) and color readers already exist for ~icrotiter plates. An automated device for carrying out the present invention can comprise: a pipetting station and n detection apparatus, said pipetting station being c~pable of p~rEorming 10 sequential operations of adding and removing reagents to the solid phase at specific time points in a thermostatted environment (i.e., temperature controlled environment). The sequential operations include one or moro of the following: mixing or 15 contacting samples, ly~is solutions and solid support, withdrawing fluid from the supports, addin~
wash buEf~r, repcating the ~tep~ ~nu~er~tad nbovo, adding labeled probe~, r~pc~tin~ thc w~sh ~top~
again, adding dotection agent~, ~nd det~cting the 20 signal with the detection apparatus.
The present prelmmobilized oligonucleotide probes and m0thod of using them can b0 incorporated inta a kit for clinical use. Such a kit would include solid supports each having bound thereto a 25 substratum, and one or more specific tailed capture probes prehybridized to the substratum. The substratum will preferably be a homopolynucleotide, such as poly~dT). The kit can also contain lysis solustions or other chaotropic agents, and a labeled 30 probe for detecting and quantifying target nucleic aclds. The kit can optionally contain wash buffers ...... .

., '' " ' ` " ' ' ` '~' '. ' i `. ` ' " ` . . . ' ' ' ' ' , .`' ' `, "'. '' ' ' ` ' . , . '' ' '. .

WO~0/10716 P~T/US~0/0~205 2 ~

a ~eans for detecting the labeled probe, one or more elution buffers, amplification or cloning reagents, and/or one or more positive control samples and one or ~ore negative control samples. Amplification oS of the target sequences can be accomplished, for example, by the technique described by Mullis in U.S. Patent 4,683,202. Cloning oE the target sequences can be nccompli~hed, for example, by the method disclosed by Maniatis i~n Molecular Clonin~
10 A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982).
The invention will now be further illustrated by the following exawplas, whlch are not intended to be llmiting in any wny.

15 EXjAMjPjLEjS .

Exjamj~le jl This example shows that laying down a poly(dT~
substratum on polystyrene greatly increases the ability to bind the preferred oligonucleotide 20 capture probes to this solid phase, which in turn improves the ability to do target capture on this support. It ifurther discloses methods for preparing multilayered substrata to increase the binding capacity of the support for the capture probes, and 25 thus, to increase the speed and efiiciency of target ~ :
capture.
':
Naterials and Methods _____________________ Co-ti-~-of-ipolystxrene-with Pol~dT) I ;
T~e following procedure is for coating i ' ' ': ' . .: ' .:' ' ' : . .. . . .. . .. .. . . .

WO90/l07l6 PCT/US90/01205 microtiter wells, but has also been used successfully for coating polystyrene tubes and dipsticks such as those produced by Hygeia, Inc.
~Newton, MA). Removawell strips were used to 05 customize the number of wells to be used per test A volume of 0.3ml of 3 OD/ml poly(dT) in 1.5M NaCl, 0.3M Trips (pH8.0), 0.5M MgC12 per microtiter well (such as Dynatech I~mulon 2) was sealed and incubated overnight at 37-C. The dT mixture was 10 then removed from the wells, which were dried at 37~C for 30 minutes. The dried wells were exposed to 650 ~W/cm of UV (254nm) for 2 minutes, washed three times with lM NaCl, lOOmM Tris (pH9.3), 2~M
MgC12, 0.1~ Tween 20, and air dried. Well~ were 15 blocked with 0.5M LlCl, 2.5~ acetylated BSA, 10 ~g/ml denatured ~. coli DNA, nnd 10 ~g/ml tRN~ ~or 1 hour at 65C, and w~shed three times ns nbovo.
Alternatlvely, 2.5~ GuSCN wns substituted for O.5M
: LiCl and the blocking was done at 37C instead of 20 65C, Determination of tjhe jBindin~ Ca~acity oi ~oly(dT) Coated Microtiter Wells To determine the binding capacity of the poly(dT) coated wells, 0.3ml of 2p 5~ end-labeled 25 dA12 (5~g/ml) (Pharmacia) in the lM NaCl wash buffer was added per well and incubated for 15 minutes at .
room temperature. The wells were washed three times with the above buffer and then scintillation counted. An acceptable dAl2 binding capacity for 30 rapid (i.e., 30-60 min) capture was:detèrmined by . .

:' ~ . .:, , . ' . :

WO90/10716 PCT/US9~/01205 2 ~ 3 ~6-experiment to be greater than or equal to 200 ng/well. ~ncoated wells (no substratum) typically had a dA12 binding capacity of less than 1 ng/well.
The binding capacity for oligonucleotldes was 05 determined by binding 32P labeled talled oligos to poly(dT)-coated and uncoated (control) wells, which were blocked as describ0d. A bindin~ mixture of 2,5M GuSCN, 2.5~ ac~ylnt~d BSA, 200~M Tris (pH7.4), 40mM EDTA, and 2.5~g/ml of the labeled dA-tailed probe was added to microtiter wells with and without dT3000 coating as a negative control. The mixture was incubated at 37C for one hour, removed from the wells (and saved), and the wells wcre washed three times with the standard wash buffor, Both ~h~ wells and the used binding mixture wero scintilla~ion counted, To dotermine thc ~mount of t~ilod probo bound to the wall~, tho following Eormuln~ worQ u~d with C defined as capacity ~ln rnicro~r~ms):

cpm added - cpm removed cpm bound C - _ Sa probe (cpm/~g) Sa (cpm/~g) ,, From the known specific activity (Sa) of the probe, the amount of tailed probe bound per well was calculated, The results of the two methods showed :
25 excellent agreement, -Pre~aration of Tailed Ca~ture Probes Oli~onucl~eotide probes were tailed overnight at 37C in O.lM potassium cacodylate (pH7,0), 4mM

.'':- ' ' :, ".'. ' '. .' ' .' ' ' ' ' ~:, ' . ' WO90/10716 P~T/US9~tO1205 -27- 2 ~ 3 MgC12, lmM 2-mercaptoethanol, O.lmg/ml acetylated BSA, dATP:oligonucleotlde at a 50:1 to 150:1 molar ratio, and 1000 units per ml of TdT (Supertechs). A : .
small amount of tritium labeled dATP was added to 05 the reaction in order to be able to determine tail length and the amount o~ tailed probe bound per well.

Results ____.___ The Salmonella specific 35mer oligo probe #676 10 (Figure 3) waq 5' end-labeled with 32p to a specific activity of 106 cpm/~g and tailed with either 30 dA
residues or 110 dA residues. The probes were bound to blank ~icrotiter wells and crosslinked with ~V as described for the poly(dT) immobilization proceduro 15 or they were hybrldized to poly~dT)-polystyrane wells in 2.SM CuSCN at 37C ~9 dcscribo~. The wcll~
which wcre not coated wlth poly(dT) bound an avorago (of five wells) of 1 ng oP the dA-30 probe and O.B
ng of the dA-llO probe, a c~pacity much too low to 20 be usePul in a rapid assay format. Surprisingly, an average of 417 ng of the shorter tailed probe and 323 ng of the longer tailed probe were bound to the polystyrene wells coated ~ith the negative control dT3000 substratum, which is significantly higher 25 than what is reported in the prior art with random sequence DNA. Suprisingly, the ability to bind the ;
:preferred.oligonucleotide capture probe to ~ polystyrene was increased 300-500x fold-by using the ~poly(dT) substratu~
~ 30 : While.virtually no dA-tailed oligonucleotide bound to untreated polystyrene using`the-prior art WO~0/10716 PCr/US90/01205 9 id ~ ~, methods, a substantial amount oi the tailed oligonucleotide probe was immobilized on the poly(dT) coated support. Typical levels of bound capture probe range form 250 to 500 n~ per well with 05 the entire ran~e producing acceptable results in assays. A lar~er difference in the amount of probe bound in relation to tail length would be expected.
A shorter tail should allow nn incre~sed amount ~f olLgonucleotide to bind, although the oligo~er with the longer tall is probably not binding to the poly(dT) with all of the nucleotides in its tail.
Increased binding of the tailed probes to the poly(dT)-coated supports has been achieved by increasing the concentration of the oligonucleotide in the binding mixture to 5.0~g/ml~lO.O~g/ml, The concept of binding a layer of poly(dT) to polystyrene to allow the bindinB O~ a multipllci~y of t~iled oli~onucleotide c~pture probos, which by themselves do not bind to polys~yrenel can be 20 further generalized. For example, tailed probes containing a multiplicity of properly spaced secondary li~ands can be bound to poly(dT) polystyrene as above. The multiplicity of secondary ligands constitute the second layer or second 25 substratum. Thus many first probes or other adaptor molecules can bind to the polystyrene and each of them would be capable of binding a plurality-of additional capture probes (potentially bound to a tar~et). In this way the capacity of the 30 polystyrene solid support can be increased to any desired level tD improve.the speed and efficiency of the capture of target molecules~ 3`'-.. - : .. ~ ~. . . . . . . .

W090/10716 ~ q.

E~ample 2 The present invention provides the same assay sensitivity as previous techniques that employ a capture probe free in solution. The rate of capture 05 with the present method is, surprising, only slight-ly slower than the rate with the capture probe free in solution. The rate of capture is thus sufficient for practical clinical use.

Materials and Methods _____________________ Coating of polystyrene with polyd(dT) and tailin~ of capture probes were performed as described in Example l.

Synthesis of Labeled Generic RiboProbe A 5' 567bp fragment of the E. coli 16S rRNA war, 15 cloned into the pGEM4 vector. (Pro~ega Biotec) It WAS transcribed wlth T7 polymarns~ usin~ b~o-ll-~TP
(En~o) according to the manu~acturer's instructions (Promega Biotec). The resulting riboprobe was then purified by two rounds of ethanol precipitation.
20 This probe is capable of hybriding through short stretches of homology to all eubacterial 16S rRNA
for which sequence information is available. It has been shown to hybridize to E. coli, Shi~ella, Salmonella, Campylobacter~ Listeria, Neisseria 25 gonorrhea, and Chlamydia trachomatis.

Bindin~ of dA-tailed Oli~onucleoti:de Probes to Poly(dT) Coated Microtiter Wells Prior to ..
~- Tar~et Capture : : -A binding mixture~of 2.5M GuSCN, 200mM Tris 30 pH7.4, 40~M EDTA, 2.5~ acetylated BSA, and 2.5~g/ml .. . . . . . .. . . .

WO9{)/10716 PCr/US90/01205 ~ ~9~ 3 30-of the dA-tailed probe was added to preblock~d microtiter wells wi~h and without (negative control) dT3000 coating. The mixture was incubated at 37C
for one hour, removed from the wells (and saved), 05 and the wells wera washed three times with the standard wash buffer. Both selected wells and the used binding mixture were scintillation counted. To determine the amount of tailed probe bound to the wells, the formula set out in Example 1 was used. ~
10 The two methods used to calculate capaci~y were in ~-excellent agreement. Control wells which were not coated with polydeoxythymidylate bound an average (of five wells) of 1 ng of tailed probe. Poly(dT) wells typically bind 300-500 ng of tailed probe, lS depending on the tail length.

Use of Pre bound Ca~ture_Probes in a Nonisoto~ic Assay Capture probes can be im~obilized ~ither on polystyrene microtiter wells, polystyrene tubes or 20 dipsticks, or other configuration, with all supports functioning equally well for assaying clinical samples. Most of the work has been done with microtiter wells, thus, the following procedure is , tailored specifically for this support, though with 25 alterations in volumes, it is easily adapted to dipsticks and tubes.
Three volumes of 1.3X processing buffer (3.25M
GuSCN, 0.4M Tris pH7.5, 0.08M EDTA, 13~ dextran sulfate, 1~ Sarkosyl) were:added ~o clinical-- -, . .. .. . .
30 samples. After vortexing for 30 seconds at top--speed, 300~.1 of sample,was added to each microtiter .. ~ . .. . . ..
. .

2 ~ L~: 3 well and capture of targets by the pre-bound probe was allowed to occur for 40 minutes at 37C.
~Capture time varies, depending on length of the dA
tail on the probe, with approximately one minute 05 allowed for each dA). The samples were then removed from the wells, which were washed with 2.4M tetra-ethyl ammonium chloride ~TEA Cl) for 15 minutcs at 37-C. This was Eollowed by the usual high salt wash. A 300 ~1 volume of a biotinylated generic 10 riboprobe mixture was added per well and incubated for 30 minutes at 37C. This mixture contained 0.5-1.0 ~g/ml ~ the riboprobe, 2.5M GuSCN, 0.2M
Tris pH7.5, O.OlM EDTA, and 10~ dextran sulfate.
The riboprobe mixture was then removed from the 15 wells, which w~re washed three time3, and the enxyme con~ugate (diluted 1:500) w~ added,to the well~
~300 ~1/WQ11) 4nd lncubated for 10 minutc~ at rooln temperature, This wa~ follo~od by thrue to 1~e washes with the NaCl wash buffer and then the ad-20 dition o~ the enzyme sub9trate pNPP (1 mg/~l) in lXdiethanolamine buffer (Kirkegaard and Perry). The optical denqity at 405nm was read when the back-ground on the negative control wells started to appesr.

Results To be a viable rapid assay method for clinical use, the pre-bound probes must function sbout ~ equally well as capture probes free in solution.
That is, the signal/noise ratio must not be signifi-30 cantly worse with the preimmobilized probe~and it must not be so slow as to be impractical in a rapid :, ... , . : . , .: , :, ,, " . , . ::: : , ,. ., ., :, . . .

WO90/10716 PCT/US90/0120s 2 ~

assay format. In order to make this comparison, Cam~ylobacter cell extracts were assayed using a pre-bound capture probe, and also with the hybrid-ization of the capture probe occurring in liquid 05 phase. A Cam~ylob_cter specific probe and the target containing 65 dA residues on the 3' end, (hereafter referred to as probe ~732 shown ~n Figure 3), was bound to poly(~T)-3000 coated microtiter wells as described above. A 300ul volume of 2.0 x lO 106 Cjampylobacter cells/ml in l.0 x 108 E. co~i/ml in 2.5M GuSCN, lO~ dextran sulfate was added to wells with pre-bound 732 probe and incubated ~or the times indicated in Figure l. As a control, samples containing l x 108 E. coli/ml, but no C~m~lobjacter, 15 were incubated under si~ilar conditions. In parallel, the same samples were preincuba~o~1 Eor on~
half hour with l ~-g/~l oE d~tallad ~732 nnd l /lg/ml biotinylatod genaric rlboprobo, and ~dded to ~, poly(dT) coated wells or the lndicated ti~es. All 20 wells were incubated or the indicated times at 37C
and then washed three times with lM NaCl, O.lM Tris pH 9.3, 2~M MgCl2, O.l~ ~ween 20. The standard assay procedure was then followed as described above for detection. Figure l shows that essentially no 25 difference in the maximum signal was observed between the assay performed with the capture probe either preimmobilized on poly(d)-coated polystyrene or free in solution. Backgrounds were substracted irom si~nals in generating Figure l and were 30 negligible in ~all cases (-less than or equal to 2~ of the maximum signal). `~
: . . .... , .. . . ; 1 . . . . .. .

2 ~ 3 A surprising result was tha~ the rate of capture with thP dA-65 cailed preimmobilize capture pro~e was only slightly slower than with the sa~e capture probe free in solution. In Figure l, 05 capture is substantially complete in 60 minutes with che capture probe-target complex free in solution and with the dA-65 tailed probe prebound to the support, Thc c~pture kinetics with both the free and the prelmmobilized tailed probe depends on the lO tail length, For the preimmobilized tailed probe, the optimal capture ti~e ;s approximately one minute for each dA residue in the tail; that ls, with a tail length of 40, 40 min i9 allowed for cnpture;
with a tail length of 90, 90 min i9 allowed for 15 capture, ~he preEerrad tail longth is about 30~40 nucleotides for quick, efEicient cnpture in 2.5 M
GuSCN.
The hlgh concentr~tlon oE praimmobilized capture probe achievcd by the present invention 20 helps to make the hybridization to the target occur quickly. The ovcrall assay times for the two formats are thus about the same (about 2 hours).
Table l presents a summary o~ the time required for each step in the method.

W090tl0716 PCTlUSgO/OI~OS
~9l~

Table 1: ~asic Assay Steps and Times Required Therefor Step Time RequLred ____________________________________________________ 05 Process samples 2 min/sample Capture with preimmobilized oligos 40 min TEA Cl stringency wash15 min Label with generic riboprobe 30 min Wash and detect nonisotopically 30 min ____________________________________________________ Note: 20 stool samples have been conveniently assayed in under 3 hours by this method. The capture time i9 that for a 40mer dA-tail on the capture probe, 15Another Pactor that is thought to contribute ~o the unexpected l~ck of di~ar~nce in c~pturo kln-etics between ~ihe ~r~o and bound c~ipture probe wlth a 65mer tail length is that prebound capture probe should still have some mobility in its interaction 20 with the target, since it is anchored to the support only through its tail. If it were nonspecifically immobilized to the solid support throu~h ~ts mixed base sequence (as are probes bound to nitro- :
cellulose), then it would have less freedom of 25 movement to interact with tar~ets.

Example 3 This example shows that there was no crosstalk amon~ dips~icks containing different prebound probes when these dipsticks were immersed in the same 30 sample. It was shown that the reason for the W~90/]07l6 PCT/US90/~1205 2 ~

absence of crosstalk is that the capture probe remains stably bound to the poly~dT) throughout the assay.
When using a number of polystyrene dipsticks in 05 the same sample, each with a different bound eapture probe, it is possible that some of the noncovalently bound probe could migrate from one support to another. Thi~ phenonamon eould result in diminished signals and/or in inereased baekgrounds The 10 following experiment was performed in order to see if such "crosstalk" occurs with this method of attaching oligonucleot~de probes to polystyrene .
supports.
A Cam~ylobacter specifie dA40-tailed probe 15 (probe ~ 732, Figure 3) and a nonseni~o sequ~nee eontrol probe (probe ~ 576, Figure 3) wer~ bound to poly(dT)-eoa~ed polystyr~ne miero~itor wolls. Th~so wells were th~n l90d ln nn ~ y in two dl~Eornnt ways: the first bein~ ns mlerotit~r w~ and th~
20 other as "dipstieks" with the entire well being submerged into a sample.
A normal ~tool sa~aple proeessed in 2.5~ GuSCN, 10~ dextran sulfate was assayed with and without the .addition of Cam~ylobacter extraet at 5.06 eells/ml.
25 Eaeh point was done in triplieate with a total of six "dipstick" wells being added to lOml of eaeh stool sample and 300~1 of sample being added to each miero~iter well. Capture was allowed to oceur for - forty minutes at 37C and was followed by a TEA Cl 30 wash (equal volumes as samples) for fifteen minutes at 37C. At this point, all of the supports were ~' ' ''' '' , ' '., . . ," ' ' . ,' ','. ' " .' ~ ' ' . '. ' ' W ~ 90/10716 PC~r/VS90~0~205 treated as standard microtiter wells and the pro-tocol described in the Materials and Methods Section was followed. The optical densiity at 405nm was read 10 and 30 minutes after the addition of the enzyme, 05 subserate. The expe~iment was repeated using the Salmonell~-speciiic capture probe 676 (Figure 3) as a control instead of the 576 control probe. In this case, the OD 405n~ W~9 read ten and twenty minutes after the addition oE the enzyme 9ubstrate. The 10 data of Table 2 shows that neither diminished signals nor any increase in background was observed when "dipsticks" with different bound probes were placed in the same sample.

Tsble 2: Absence oP "Crosstalk" Among Dipstick3 OD 405nm OD 405nm SampleSupport Probc 10 min. 30 ~n.
unspiked'stooldipstick 732 0 0. 044 " 576 0 0.041 st~iool * c~mpy " 732 1.474 2 0 20 " " 576 0 0 030 unspiked stoolwell 732 0.004 0.064 ~ 57~ 0,009 0 060 stool + ca~py " 732 0.529 1 226 25 " " 576 O 0.060 :
20 min unspiked stooldipstick 732 0 0.014 ~ " 676 0 0.0~6 stool + campy " 732 1.254 2 O
30 " " 676 0 0 009 _ unspiked stoolwell 732 0 0.014 stool +-campy '' 73726 0.584 1 133 -" ; " 676 - 0 -- 0 017 Legend. Filtered GTS nor~al s~ool #14 in 2.5M

,`,' ~ ' ~ ` ' ` .' . . ~, . ` ' '.

WO90tl0716 PCT/US90/01205 GuSCN, lO~i dextran sulfate was spiked with Campylobacter extract at 5.0E6 cells/ml. In the firs~ experiment an average of 556ng of 732 and 05 316ng of 576 were bound per poly(dT) coated well, and in the second, 445ng of 732 and 350ng of 676.
To each well was added 300ul of sample and six "dipstick wells" were added to lOml of sample.
After the TEA Cl wash (in volumes equal to those of 10 the samples), the "dipsticks" were treated as normal microtiter wells and the standard protocol was followed. Results are the average of triplicate samples.
As compared to the results from the wells, the 15 backgrounds from the "dipsticks" were slightly lower (not significantly hi~her, as predicted if crosstalk occurs) and the positive signals were actually greater (not less, as predicted if crosstalk occurs). The increasc in signal seen with thc wclls 20 used as dipsticks could be due to the largcr snmplo volume available to the ~ells im~orsad ln tha samples couplcd with tar~et drivon hybridix~tloll at this level of t~rget. In any case, the higher slgnals achieved with the actual immersion of the 25 solid phase a9 compared to the use of microtiter wells is one advantage of using dipsticks as the solid phase in place of microtiter wells. Another advantage of dipsticks over wells is that sample handling and pipeting are minimized.
~ost likely, the absence of crosstalk is due to the tailed proba's remaining bound to the support throughout the capture. Additionally, if a small amount ~f loosely bound tailed probe does elute from a gi~en dipstick, very little can rebind to other - dipsticks since the vast-majority of the poly(dT) on ., ., , ! :

:: . : . "' ': ' ~ . , ::' :
, :' : ' . ' ' ' , . ': . . .
' , " ' ' :: :
:. ' ~ " ' " ~ , . ,. ' ~' ; . . ' '. ~' ~, ' ' ~ V~ -38-the other sticks have been presaturated w~th their own tailed probes. An experiment was done in order to measure the stability of the prebound tailed probe during the course of the assay. The tritium 05 labeled, dA-tailed capture probe 732 ~dA80) was pre-bound in 2.5M GuSCN, 10~ dextran sulfate to dT4000 coated and blocked polystyrene microtiter wells with ~n average dA12 binding c~pacity of 412ng. Tw~lve wells w0re scintillation counted 10 prior to the assay in order to establish a baseline for the amount of probe immobilized on the supports.
The results are presented in Table 3. A mean value of 274 ng of probe bound per well was obtained.
Twenty~four wells were then carried through ~ mock 15 assay which included all oP the normal st~ps and reagents, excopt that no t~rget or riboprobc waro ~dded. Twelva well~ wcre ~ny~d wlth tho ~tnndnr~
2.5M GuSCN, whilc the other twelvo wcra dono wlth 2.0M GuSCN. 2.0M GuSCN was u~od because it effect-20 ively controls RNase, and oligo(dA) binds topoly(dT) bett~r in 2.OM GuSCN than in 2.5M GuSCN.
The wells were incubated with Z.SM or 2.OM GuSCN and lO~ dextran sulfate for 40 minut0s 37C, followed by a 2.4M TEA Cl wash for 15 minutes at 37~C. After 25 three washes with lM NaCl, O.lM Tris pH9 . 3, 2mM
MgC12, 0.1~ Tween 20, the wells were incubated once -again with GuSCN and 10% dextran sulfate for 30 ,-~
minutes at 37C. Following three washes with the above NaCl buffer, the wells were incubated with the 30 enzyme conjugate dilution~buffer for 10 minutes at room temperature. The wells were washed three times with the NaCl buffer and then scintillation counted.

,~ . .

- . , WO90/10716 PCT/~S90/01205 2 ~ 3 (The enzyme substrate step was excluded since it may actually be advantageous i the probe came off of the sold support during color development). As seen in Table 3, a mean value of 198 n~ of probe retained 05 per well in those which were assayed with 2.5M
GuSCN. Based on the pre-assayed values, 72% of the immobilized probe remained boutild to the solid supports. For the wells assayedi with 2.0M GuSCN, the mean value retained was 218ng per well, or 79~
10 of the established baseline. This shows that very little capture probe comes off the support durin~
the course of an assay wlth the present invention.

.. - ~, . . .

2 ~

Table 3: Retention of Prehybridized Capture Probe on Poly(dT)-Polystyrene During the Course of an Assay rr~ s~ ;u~ s.;:~ 0'1 GuS''`; ~:S~
05 ~ ~ ,n~ 1 n~ /t~ n~ tt~ l l n~ nl/t;
___________________________________________________ _________ i~G2~ ~3 1~l10 l~ 0 1l3 ~10~G 301 ~0~ ~00 ~-l5~3 ~0~
~fila 2~1 13~30 1~ 1 R3l ~ 5 ~136 3~0 ~f.0~0 ~29 ll~no ~o 10 110~7 213 11_69 1fi113~.l0 1.J0 ~9~93 "8~ ~3131 ~92 1~93 1 ~-ll ,l1 1-,/0~ ~10 lC~
1l89~ ~5~ 213 1~0~0 ~3 ~105~ 301 15031 215 1l-l39 l~ 20~0~ 91 13~1 190 '170n~ 2l3 lG60~ 237 1391~ 199 11300 ~0~
18fi80 2~/ ;26;,8 11 1~9/ 193 ____________________________________________________________ , me~n: 27~ 198 218 s~d. de~ 8.3 1~.3 ~7.
~ ~O reteined: 72,' l9~O
____________________________________________________________ Le~end: Pol~stS-rene mi-roti~er ~ells ~ere co~ted ~:ith ' pol;(dT)-tO00, ~' irrac_zted, and blocked as described. The ¦ 3ii-labeled, dA-tailed c~pture probe l32 (dAR~ ;25 bounfi to I 25~-ells in 2.~1 GuSC.~, lC~o de~:trzn sulfate. T-;el~e ~-ells ;ere scintillation counted prior to the start of the assa~- in order to obtain a baseline of th~ amount of probe bound. A -mocl~ assaS- ~no t~rset or riboprobe added) ~;as done ~ith 21 -~;
;ells, 12 of ;hich ~;ere used ;ith 2.~:1 uUSC.~ and l'> ~ith t 0 305uS~ fter thè final ~ash step, the ~;ells ~ere scintil}ation counted ~nd the percent of the oapture probe ret~ined on the ~ells has c~lculated.

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wo so/ln7l6 PCI/US90/01205 2 ~

The reten~ion of the preimmobilized probe is thus about 70-80% during the course of the entire assay Crosstalk is most likely to occur, if at - all, during the first forty minute capture step.
05 During the capture step, retencion of bound capture probe is greater than 93%.
It is also possible to add PolytdT) to the samples to bind to the dA-talls of any probes that elute from their respective dipsticks. This would 10 prevent the dA-tailed probes from binding to a small amount of unhybridi~ed poly(dT) on other dipsticks (by choosing the appropriate concentration of poly(dT), the solution phase hybridiæation will be faster than the solid phase immobilization).
Poly(dA) could be added to the samples in place of poly(dT) in sufficiont quantity such that any free poly(dT) on thc dipstlck~ will profer~nti~lly bind the added poly(dA), ~n~ no~ thc t~ d probo~
from other dipstlcks.

20 Ex-m~le 4 This example shows that the pr~sent invention can be used to assay clinical stool specimens nonisotopically with no cell culture and without prior purification of the nucleic acids. The 25Sensitivity and specificiey are each about 90%.
In two separate experiments, a total of 32 Cam~ylobacter culture-positive stool samples (obtained from Evanston Hospital, Evanston IL), and 20 "normal" and culture-negative specimens, were 30assayed using a pre-bound C_m~Ylobacter specific 16S

~VO90/10716 PCT/~S90/01~0S
3 ~ ~

rRNA capcure probe (~732). The 732 probe is 35 nucleotides in length and recognizes C ieiuni, C.
coli, and C. la~idis, but misses all other Cam~lobacter species, includin~, C. fetus and C.
05 ~ylori The standard assay protocol described in the Materials and Methods section of Example ~ was used. The OD 405nm was read 45 minutes af~er the addition of the enzymo substrato. For each sample, the level of specific hybridizntion was computed as 10 the OD 405nm of the 732 probe well minus that of the control well containing only calf thymus DNA. The cut off for positives was set at twice the average of the negative samples assayed in each experiment.
Table 4 shows that of the twenty negative stool 15 samples screened, three were scored as fal5c positlve (85~ specificity). Bettor spociELcity cnn be achieved by using two spociPic probe~ in plac~ oE
one specific nnd ono gonoric probo, S:L~n:L~lcantly, two of the three Palse positiv~s were from "normal"
20 samples, some of whom may have an asymptomatic low level Cam~ylobacter infection from prolonged exposure to the pathogen. These samples were not confirmed culture negatives. With confirmed culture negatives, the assay showed one false positive (90 25 specificity) ~, `' ' ` ' . ~ ' .,.. ! . ~ ~, . .

wr) 90/10716 PC!/US9û/0120~
2 ~ ~ 3 ~ ~3 Table 4: Assay of Normal and CulCure Negati~te Stools with the Cam~lobacter Probe #732 n~,.....
Saml-le 73" CT 732-CT ~/-05 E:~:r)erimenl ~ )rm;ll st.ools from As~mptonl~t;c Cene-TrFIl;
Donol~s n.~o~ o t).~OC
2 0 . 006 () t) . 00~ -3 0. 122 0 0. 122 +
10 ~ 0,035 0 0.()35 0 . 0 3 (~ () O . 0 " () 6 0 . 072 0 . ()~.8 () . 04~1 -7 0 . 05~ 0 0 . 0~2 8 0.078 0.031 0.0~ -.. 5 9 0 0 0 I 0 0 . 028 0 0 . 028 Cu~ off for positives: 0.091 ~2~: lhe ~ r~ of ncs.'~i. ) ____________ ______________________________________,~________ E,~ eriment ~: Glinic~l Catnp~lobacter cul~u~-ne~ati~
stools .
~0 ZO~7 0. 107 0 0. 107 Z098 0.015 0 ().095 Z05~ 0 0 0 Z06 1 0 . 07 1 0 . 00 1 0 . 070 Z065 0.124 0.015 0.109 Z0~0 0.219 0.441 0 Z081 0. 391 0 . 032 0 . 359 +
7 083 0 . 01.9 0 0 . 019 Z084 0. 09~ 0 0, Og:~ _ .
Cut off for positi~es: 0.160 (2~: the ~-g. of ne~s. ) ______________~______________________________________________ ..
Legend: See Table 5.

, . . ; , ,. . . . . ~ . .. ,, -, ", 2 ~

Table 5 shows that twenty-nine of the thirty-two Cam~ylobacter positive clinical samples proved to be positive by the DNA probe assay (9l~
sensitivity). The three false negatives, which all 05showed absolutely no signal, may have been missed due to the somewhat limited inclusivity of the capture probe used. It is also possible that the : samples were "mishandled" prior to assay since they were not collected and stored with an assay for a lOfragile molecule like RNA in mind.

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Table 5: Assay of Cam~ylobacter Clinical Samples :-~ 105r~m Sanlp1e ,~2 CT ~32-CT ~/- CuI~ure E~:periment i~: Cut, Orr for p()~ es: 0.09-1 057.00~ >~ 0 >.
zoor, 1. /2 n 1.2,2 ~ ~P
Z00~ ~2 0 )2 + 3P
Z0~7 0.()00 0 0 Z008 O.n11 0.0~3 0 - 2 L OZ009 0.1~9 0.051 0.3~5 + _P
Z010 ~2 ~.3~3 ~O.G t 1~
Z011 `2 0 >2 + 3P
Z012 `~ ~.l8~ ~1.2 + 3~
7013 0.~2~ 0 0.~ + ~P
15ZOI~I >~ 0 >2 + ~
2015 >2 0 >2 ~ 2P
Z016 >~ 0 `2 + 3~
Z017 >2 0 :~2 t lP
Z018 0.097 0 0.097 ~ iP
20Z019 ~2 0 )2 ~ 3P
Z0~0 ~2 0 >2 ~ ~ 3~
Z021 >2 0 >~ ~ IP
Z0~2 0 0 0 - 2P
Z023 >2 0 >2 * 2P
252024 0.~8~ 0 0.4~6 ~ .~P
Z025 >2 0 ~2 +
Z026 0.128 0 0.1~8 + lP
Z027 0.~1~ 0 0 416 + 2P
Z050 1.028 0 1 0~8 + ' 30Z062 >2 0 >2 +
E~;periment B: Cut o~f for positi-~es: 0.160 Z06~ >2 0 )2 + `
~090 >2 0.1~?8 >1.8 + 4P ~:
Z091 >2 0 >2 + 4P
35Z09~ >2 0 ~ p Z100 ~2 0 >2 + - 3P :~
Z112 >2 0.0~0 ~2 + 3P
________________________~____________________________________ Legend o~ Tables 4 and ~: The "/32" column is the Camp.~~lobacter specific signal and the "CT" column is the ~ ~0 nonspecific ~czlf th~mus DNA) bacl.~round. ~/-refers`to ~hether the sample i positi~e or ne~ati~e b~ the D~'~ probe assa~:. Culture: 4P ~> 3P 2P )j 'P. This represents a relati~e estima`te of the number of (:amp~lobacter colonies on the ~ selecti~-e plate.

2~ 4~ -46-Exam~le 5 This example shows the detection limit of the assay by measuring the dose-response of the tech-nique for the detection of Salmonella ty~himurium in 05 25% w~v normal stool.
In order to assess the de,tectLon limit of the ; assay described in this document, a dose-response study was don~ for ehe direct detection of Salmonella spiked into normal stool. Normal stool ; l0 was spiked with various levels of Salmonella ty~himurium and assayed following the standard protocol of example one but using the Salmonella specific capture probe #676 (Figure 3). As shown in Figure 2, the nonlsotopic assay detection limit 15 (without cell culturo or prior puri~ication of thc nucleic acids) is ~pproximntaly l.2 x 104 Salmo_oll~
per sample. Tho sign~l at th~t lavol oP 9almon~ln (0,038 OD) is about l0 timos tho ~ssny b~ckground (0,004 OD).

20 Example 6 This example shows a useful, generic method whereby a stringency wash can be done with target immobilized on a solid support by means of the poly(dT)-poly(dA) affinity pair.
The wash step with 2.4M TEA Cl following ~capture of tar~ets out of samples was originally added as a stringency wash in order to reduce nonspecific hybridization. 2.4 M TEA Cl functions as an agent that equalizes the strength of the AT
30 and GC base pairs. This.property can be exploited in assays employing a battery of probee of a fixed ........

W09~ 7l6 PCT/US90/01205 -47 ~ a~ 3 length but of widely different base composition. By washing with 2.4M TEA Cl, all hybridizations can be made equally stringent. In this way a reduction of nonspecific hybridization can be accomplished with a 05 TEA Cl wash.
Tetraalkylammonium salts have an added ad-vantage for a capture system ba~ed on the dA-dT
sffinity pair: They greatly stren~then the poly(dA)-poly~dT) affinity pair (dissociation 10 temperature of 65C in TEA Cl) relative to 35mer mixed base sequences (dissociation temperature of 43-44C). This homopolymer thus enjoys an un-expected but useful stability. (Because of the equalizing of G-C and A-T base pairs in 2.4 M TEA
15 Cl, a plot of dlssociation temperature ~orsus probo length is a straight line. Wood et al. Proc. Natl.
_cad._Sci._US_., vol 82, 1585~1588 ~19n5). Thls l~
true Por random mlxod b~e ~oquances. Poly~dA)-Oligo~dT)-14 and poly~dA)-poly~dT) have dissociation 20 temperatures much higher than predicted by their method.
Because of the special stability of poly~dT)-poly~dA) in reagents such as TEA Cl, a lot of pressure can be applied to dissociate the probe from 25 pseudo-target sequences containing only one or a few mixmatches. The hybridization background can be greatly reduced under such washing conditions. The dA-dT pairing would normally break up under such stress and most of the signal would be lost with the 30 background if it were not for the special and surprising strength imparted to ~he dA-dT homo-polymer in reagents -such as tetraalkylammonium salts.

W090J107l6 PC~/US90/012~5 f~ ~J~ Li ~

Exam~le 7 _ _ _ _ _ _ _ _ This example shows some further unexpected results obtained with the TEA Cl stringency wash.
Another surprising result was the reduction in 05 nonspecific binding of the labeled probe to the support by washing the support PRIOR TO the addition of the labeled probe (TAbles 6, 7, and 8). Whil~
the reduc~ion of nonspecific hybridizstion of the probe to pseudo~targets is expected with TEA Cl, the 10 reasons for the reduction in nonspecific bLnding to the solid support are not completely understood.
While in relatively clean samples the addition of the TEA Cl wash sometimes has little effect on nonspecific backgrounds, it does reduce the higher lS nonspecific backgrounds encountered in very dirty samples. The lower backgrounds in Table 6 seen with the samples wnshed wi~h TEA Cl mu~t be du~ to th~
decrcased nonspeci~ic binding o~ ~h~ l~belod probo to the solid support since the control which has no 20 heterologou9 target i8 the same as the control that has heterologous target. The lower "E. coli"
isotopic background seen in Tsble 8 must also b~ due to a lower pure nonspecific b~nding of ~he riboprobe to the support following a TEA Cl wash (there is far 25 too little homology between the Cam~ylobacter probe and E. coli 16S rRNA to form stable hybrids in 2.SM
GuSCN at 37~C).
Another surprising result obtained from the use of reagents such as TEA Cl for washing was the 30 increase in signal when the wash step was done prior to the riboprobe hybridization. Wash steps do not normally increase hybridization signals. This can W O 9~/10716 PC~r/US90/01205 3'~
-49.

be seen in the results presented in Table 6, where the inclusion of the TEA Cl wash effectively doubled the signal from the Campylobacter sample (Cam~lobacter specific capture probe 732 used).
05 The low nonspecific barkground from the E. coli and no cell control samples were further reduced by the TEA Cl wash.

Table 6: The Vtllity of tht~ TEA Cl Wash in a Nonisotopic Assay 0,on 1 0 ."~
~tlll:lt.' ~r~A ~ ,r7~ lG
____________________________________________________________ Camr,~lobact~r ~ 4 0.()16 0.
E. coli - 0~035 0. n~o !).
:~o cells - 0.021 0.015 0.~0 ___________________________________________________________ ~
Canlp~lo~uctcr t 1.1~,l 0.0"1 1.1~6 . coli ~ o.nn6 ~.~18 0 rlo cells t Q.011 o,oot~ 0 ____________________._______________________________________ Le~e~lt~: C:lnlp~,lt~buc~er ~pec:iric 732 cnp~:ure probe wuP pre-~ound ~o pol~ (dT) pol~st~rene ~ lls. Ccll e~;~ructs l~er~ ~ilu~ t.~ in 15 2.o~ CuSC~, 10',~ de~;trull sulfate to l.OE~ Camp~lobacter~ml or 1.0~ ~. coli/nll. Af~er the capture stt:~), the l~ells ~ere ~mp~ied and ~ A Cl ~ c. added to ~he indic~te~ ~;ells ;~lld incubated ror 1~ minutes at 37 de~rees. All wells ~ere 1.hen ~shed ~; in the lM ~'aCl w~sh buffer an-~ ~he ~ss~ ;as 20 con~inued usin~ the stal~dard ~ro~o~,t~l. The on ~ nm w~c. read 30 minu~es æft.er the addition of the enz~-nle su~stra~e. The result~ are ~he a~era~e of duplicate samples.

An experiment was done to deter=ine if TEA Br producted the same eff~cts in the assay as does TEA
25 Cl (Table 8). TEA Br does cause a lar~e increase in si~nal when used in a wash step prior to riboprobe hybridization. The TEA cation is thus responsiblP

.

3 -`

for the effects, although this does not imply that the anion is completely immaterial. Other cacions, particularly similar cations, should have the same effect.

05 Table 7: Comparison of TEA Cl and TEA Br Washes ~D 40r~nm S~mr)l~ Wash 20 min, ~0 min.
__________________________________________________________ __ unspiked stool - 0.0~3 0.0~7 stool t Camp~. - 0.082 0.183 0l~nspil~ed stool T~A Cl ~.030 0.0~
stool + Camp~. TEA Cl 0.525 1,620 uns~iked stool TEA Br 0.0~1 0.059 fitool ~ Cam~. TEA Br 0.-~8 1.~04 _________________________________________________________ Le~end: Normal stool was assa~ed h'it}l and ~ hout ~he lS addition of Camp~lobacter e~tract to 1.0EG cells/ml.
1`he standard ass~y protocol wa~ follol~ed with the ~.~a~ption of t,he TEA Cl wash ~tep, which wa~ vuried. In one,s~t o~
camples it w35 includ0d, in anoth~r it was replace~ wi~h TEA
Br, and in another it was also e.~cluded. Both the 2.~1 TEA
20 Cl and 2.4~1 TEA Br ~ere autocla~ed prior to use. The OD
~05nm was read 20 and 60 minutes after the addition of the enz~me substrate. Results are the average of duplicate samples.

Another important aspect of this experi~ent is :~
25 that about an eight-fold increase in signal was seen ~,~
(with both TEA Cl and TEA Br) instead of the ~two-fold increase previously observed. This could be due to the fact that both reagents were autocla:ved before use, which may have eliminated a 30 low level nuclease activity which was previously present (and which thus may have lessened the potential signal enhancement).

.3 Table 4: Assay of Normal and Culture Negative Stools with the Ca~ylobacter Probe #732 Sam~le 732 C~ 732-C~
05 E~;~erimenl. ~: N'ornl~l st.ools from As,~n~ptom~tic Cene-Tr~li Donol ~i I O.~OG 0 O.~OC +
2 0.006 0 n . oo~ -3 0.1~ 0 0.122 ~ .`
104 0,0~,5 0 0;035 0.0~0 0 o,o~ _ 6 0.072 0.~8 ().O~;l -7 0.052 0 ~ ~
8 0.078 0.031 0.0~ -'.59 0 0 0 0.028 0 0.028 Cut off for positi~es: 0.091 ~2~; lhe ~ . of ne~
_____________________________________________________________ E~eriment B: Clinical Camp,~lobacter culkure-n~ti~
stools.
~OZO~7 0.~07 0 0,107 Z0~8 0.015 0 ~.0 Z05~ 0 0 0 Z061 0.071 0.001 0.070 - ::~
Z06~ 0.124 0.01~ 0.109 25z070 o 0 0 Z0~0 0.219 0.441 0 Z0~1 0.391 0.032 0.3~9 ~ `
~083 0.019 0 0.019 - .
. Z084 0.095 0 0.09~ - ;
: 30 Cut cff for positi~es: 0.160 (2~; the a~-~. of ne~s.) _________________________ ____ ____~________________________ Legend: See Table 5. :~
. . ~ :

... :

i . .

Wo9~1071~ PCT/VS90~0120~
~3'~3 :-`
, -50-b for the affects, although this does not i~ply that the anion is completely immaterial. Other cations, particularly similar cations, should have the same effect .

05 Table 7: Comparison of TEA Cl and TEA Br Washes ~D 40ronm Sam~ Wash ~0 min. ~0 min.
______________________________________ ______________________ unspiked stool - 0.023 0.047 stool + Camp-~. - 0.082 0.183 ~nspiked stool TEA Cl ~.030 0.0~5 stool + Cam~. TEA Cl 0.525 1.620 uns~iked stool TEA Br 0.02~1 0.059 st~ol + Cam~. TEA Br 0.~R8 1.504 ___________________~w_____ ______________________________ ~
Le~end: Normal stool h~as assa~ed with an~ hout th~
15 addition of Camp~lobacter e~traot t~ 1.0~G cells/nll.
The s~andard ass~ protocol l~a~ .îollowed with th~ e.~;c~ption of ~,he TEA Cl l~ash ~tep, which wa~i v~r.ied. In on~ ~et Or is.amples it w~s inoluded, in ~nother it wai~ r~placed wi ~h l'E.
Br, and in another it was also e~cluded. Both the 2.~1~1 TEA
20 Cl and 2.qi~1 TEA Br were sutocla~ed prior to use. The OD
~06nm was read 20 and 60 minutes after the addition of the enz~me substrate. Results are the avera~e of duplicate sa~ples.
...... _ ,,, , _ _ _ Another important aspect of this experiment is 25 that about an eight-fold increase in signal was.seen (with both TEA Cl and TEA Br) instead of the two-fold.increase previously observed. :This could be due to the fact that both reagents were -~
autoclaved before use, which may have-eliminated a 30 low level.nuclease~activity which was previously-present (and which thus may hav& lessened the -potentlal signal enhancement~. -WO90/~0716 PCT/US90/01205 -5l- 2 ~ ~ J~

In order to determine lf the increase in signal seen with the use of reagents such as TEA Cl and TEA
Br as wash buffers was due to i~proved binding of the riboprobe to the target or to a long range 05 effect on alkaline phosphatase activity, an assay - was done using a double labeled ( P and biotin) riboprobe. Samples were assayed using the - Campylobacter specific capture probe 732, with and without the inclusion of the TEA Cl wash followin~
the standard protocol. The OD 405nm was read 40 minutes after the addition of the enzyme substrate and as expected, the Cam~ylobacter positive aignal was approximately doubled with the inclusion of the TEA Cl step (Table 8). Following color development, the wells were washed and then scintillation counted to determine the amount of riboprobe bound to the wells by way of the 32p label. Approximately twi.cn the number of counts were obtnlned in the Cant~ylobacter wnlla which rocnivnd tho TE~ Cl wnDh versns those which did not ~The TEA Cl used in thia experim~nt was not ~tutoclaved). This corrospond~ to the increaae in color obtainnd with the TEA Cl wash step, indicating that this improved signal is due to an increase in ~iboprobe binding. This increased binding of the riboprobe is believed to represent increased hybridization.

, 2 ~ 52-Table 8: Effect of TEA Cl Waisih on the Subsequent Binding of the Riboprobe to the Target/
Capture Probe Complex Sample TE~ Cl OD 405nm CPrl _________ _______________~__________________________________ u~Cam~--. - 0.~96 92~
C~mp~. ~ 1.073 ll53 E. coli - 0.089 211 . c~li + 0.071 5 ____~________________________________________________________ Le~ d: Cell e~;tr~cts were diluted in 2.~M GuSCN, ~0%
tOde~tran sulfate (Cam~-lobacter to 1.0E6 cells/ml and ~. coli to l.OE9 cells/ml) and tar~ets were captured with pre-bound ,3~ probe for i~O minutes at 37 de~rees. The wells l~ere washed ~ith either ~M NaCl or ~ith 2.qM T~A Cl ~or ]5 minutes at 37 de~rees. The tar~et was then lab~led ~ith a +~. +
1516S r~'A generic riboprobe l;hich was dc,uble labeled with i biotin and 32P. ~fter oolor de~elopment with S~-AP/p.~'P~ for ~O minutes, the wells l.~ere washed and the amount of 32P bound was deterrnin~d by scintillation countin~.

While a slight dacreas~ in ~he background from 20 the jE. coli sample was observed upon color de~elop-ment when the T~A Cl step was included, a far greater decrease (4.5x) in the a~ount of riboprobe bound to the wells was seen from the 32p counts obtained. This 4.5x reduction in background in the P counts represents the true decrease in the level o~ nonspecific binding of the riboprobe caused by the TEA Cl wash. The failure to see this same level of reducti~n in color is probably due to variable nonspecific binding of the enzyme conjugate 30 streptavidin-alkaline phosphatase.
The data of Tables 6, 7, and 8 indicate that when the TEA Cl wash step is applied prior to the , W090/107l6 PCr/US90/01205 addition of the labeled probe, the level of subsequent binding ~presumably hybridization) of the labeled probe to captured targets is increased by factor of two or more.

05 Exa~ple 8 This example shows methods whereby the prescnt probes c~n bc used to do reversible t~rgct capture.
The ~dv~ntages o a reversible target capturc protocol, particularly greatly increased sensitivity 10 and specificity, are ful}y described in Collins, European Patent ~pplication Number 265244, which has been incorporated herein by reference.
One i~portant advantagc of the dA-dT afEinity pair used to capture the probe~target complcx onto 15 the solid support for the pr~9ent invention is thnt it i9 r~adily rcv~rsibl~. Revorsibillty oE tnr~qt capture nllows rccapturo o~ tho t~r~ct/probe complex onto other solid supports in ord~r to reduce back-ground levels and thus to improve the sensitivity 20 and specificity of the assay. While the preferred user-~riendly embodiment of the assay we describe in this document employs only a single capture for ease of use and yet has very good sensitivity and specificity in clinical stool samples, increased 25 sensitivity and specificity may be gained by mul-tiple rounds of capture. The experiment described below demonstrates the feasibility of the first such - reversibility scheme. This method would use a capture probe vith a very short, chemically syn-30 thesized dA tail, in this case dA15. The probe would be immobilized on a poly(dT) coated support in . . ,:

2~ 54-a tetraalkyia~monium salt such as but not limited to 3.0~ tetramethylammonium thiocyanate (TMA SCN).
Tetraalkylammonium salts impart special stability to the poly(dA)-oligo(dT) affinity pair. Following the 05 capture of the target from the sample and subsequent washes, th0 probe-target from the sample and sub-sequent washes, the probe-tnrget complex would be eluted form the solid ~upport in 2.SM GuSCN and then recaptured with a second probe with a long d~ tail 10 (either with the same or with a different mixed base sequence) on a second poly (dT) support with the same physical composition or with a different composition. As seen in Table 9, high levels of probe 88S-dA15 (Figure 3) bind to poly (dT) coated 15 polystyrene in 3.0M TMS SCN, but not in 2.5M Gu SCN.
This shows that targets can be captured in 3.OM TMS
SCN; then the probe-target complox cnn bo eluted with 2.5 M ~u SCN to allow subscquant rounds o~
capture with a second probe contnining a longer tail 20 such that the probe-target complex can bind to the poly(dT) in 2.5 M GuSCN. (The concentration of the 888 probe required for acceptable levels of binding to the poly (dT) was increased from the standard concentration of 2.5 ug/ml due to the fact that a 25 crude prep of the oligo was used in-the immobiliz-ation procedure).
- - - . .

.

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' . '`:: ' ` :~' `: .; . . .;, . ' ' ::: ' : , ', : ' '" ' : ' ' ` ` ' WO90/10716 PCT/I)S90/01205 -55- ::

Table 9: Binding of dA-Tailed Probe (dA15) to Poly~dT) Coated Polystyrene Wells in :~
Gu SCN and TMA SCN

rid. Sol. Prohe Conc. ng ~ound/well _______________________.____________________________________ 05 2.5M Cu SCN 2.i~ ug/m] 28 3.0~I T~IA SCN 2.5 u~/ml ll9 3.0~I TMh SCN 5.0 u~/ml 374 3.0M TM~ SCN lO.0 u~/ml 73~
____________________________________________________________ Le~erId: 32P labeled 6'dA-tailed 888 (dAl5) probe was bound lO to pol~ (dT) coated ~ol~st~rene microtiter ~iells in 2.5i~I Gu SCN or 3.0~ Ti~lA SCN with ~0% dextran sulfate for 1 hour at 37 d~rees~ e]ls ~ere ~re-blocI~ed wi~h the st~indard blockin~
buffer containin~ 2.5M Cu SC~' or with substitution b~- 3.0i~I
T~IA SCN. ~ollowin~ the incubation p~riod ~ith the probe the 15~i;ellS l~ere ~ashed three times in the st~ndard I~I ~aCl ~ sh bl~ffer and then scintillation counted to det~rmine th~ amount ~f probe bound. The d~ta sho~ tha~ this short 5' tailed probe will bind to poly~dT) in 3.0 ~ T~l~ SCN but not in 2.6 ~I
Cu SC~I. The la~ter can thus be u~d as an elu~ion huff~r.

20 EXami~le 9 This example discloses methods employed by the present invention to mini~ize signal loss due to the presence of large amounts of heterologous rRNAs endogenous avidin other biotin-binding molecules 25 and other compounds.
When specimens are being tested-for any of-a group of pathogens it is desirable to use a single labeled p~obe to label all of the targets of interest. For example for a GI panel a single 30 large labeled probe (such as the 5' or 3 16S
riboprobe? containing many detectable ligands;that .. . . . .

W~90/10716 PCT/~S90/01205 iJ ~

would label the 16S rRNA of Campylobacter, Salmonella, and Shi~ella would be desirable in place of a vast multiplicity of singly or doubly labeled oligomers which would have to be made for each of 05 the organisms (to achieve the same number of reporter groups bound per rRNA target), Howeveri in a s~ool sample there are generally more th~n 10 bacterla/ml. Kuritza et al.
A~lied and Environmental Microbiolo~y, 51:385-390 10 (1986). These bacteria would contain more than 1015 16S rRNA per ml, and in a competitive situation, perhaps only 1/1000 rRNAs of the target organism , will be labeled since the labeled probe COllCerltratiOn i5 only 1012/ml. This can be 15 corrected by speclfically c~pturing tho t~rgct out of the stool sample ~nd w2shing ~w~y compctltor rRNAs prior ~o l~belin~. , Various ~ub~tnnccs be~idos r~NAs pres~nt insido cells can interPere with the l~beling and detection 20 of nucleic acid targets. For example, a bio-tinylated probe was used in the experiments reported herein. If large quantities of avidin or other biotin-binding macromolecules were present in the sample, these could bind to the biotinylated probe 25 and reduce the size of the signal generated by the streptavidin-alkaline phosphatase. Capture of the target prior to labeling as disclosed herein is a - ' workable solution to this problem.
Another potential interference is endogenous 30 poly(rA) and poly(dA). Mammalian cells, which are' -found in virtually all'clinical samples'(especially blood) contain,about liattomole''of poly(rA) per'' ' -, : :: :: : .: - : . : : :~ : : :

W090~0716 PCT/US90/01205 ~Li~3~

cell. T. Maniatis, et al. 1982. Molecular Clonin~.
A jLaboratory_Manual, p. 188, Cold Spring Harbor Laboratory, New York. They also contain about 100 ppm poly(dA) by weight. These could compete with 05 the dA-tailed probes potentially bound to targets for binding to the poly~dT) polystyrene. The result would be dlminished signals and potentinlly higher '' backgrounds as well. By presaturating the support with tailed probes, this potential competLtion could 10 be minimized or eliminated along with any potential increase in background.
In Table 10, data are presented from two representative experiments compnring the results employing either the preimmobilized cnpture probe or 15 the cnpturo probc ~ree in solution, Three situations nro considarcd: no competitors prosont (5X106 Campylobnctnr/ml only), comp~ltor rRNA
present ~purified E. coli RNA ndde~), or 5 treptavidin present at 10 ~/ml. With the capture probe free in solution, there is a substantial 1099 o~ signal with competitor rRNA (34-fold decline) and streptavidin ( 100-old), as predicted above.
With the preimmobili~ed capture probe, however, there was no loss in signal with competitor rRNA or 25 with streptavidin. Thus this method allows us to exploit the advantages of using a single completely generic detector probe that ià conveniently labeled with moieties such as biotin.

WO gO/10716 PCr/US90/01205 2~
-sa-Table 10: Comparison of Competitor Effects on Solution and In~mobllized Assay Formats ml71 e OD 1 (h~nm ~)LI~r ] o~l ] ~ o~ I 1,1 Zl I) _______________________________________________________.____ 05 E~pimcnt A:
Ci~m~ b~ctel l.209 l.O~
Camp~lo~acter ~ ~, coli R~ 0.033 1.226 C~m~lo~oter ~ Strop~a~idin 0.000 1.398 ________________________________________________,____________ E~pcriment B:
amp~lobacter 0.9G~ 9~
Ci~mp,~lobacter + ~. coli R.~'A 0.03~ 0.1~9 Camp~lobiucter + i~trepta~idin 0.006 O.G25 Le~end: Camp~lobacter specific capture probe t3? (d~63) ~as used to assa~ Camp~lobacter e,~tracts ~5.0E6 oells/rnl~ in 1~ presence of E. coli ~.~A (appro~. 100 u~/ml~, pol~A) ~lO
u~/ml), or ~trc~ptiu~idin ~10 u~/ml). Poly ~dT) coated polyst~r~ne dipsticks w~re u~t~d ilS tl solid supp~r~ ~'or thc c~pture of tar~et molcoul~. Two difr~r~nt ~ssn~ form~ts were used. I~ thc fir~,t, dip~.tioks l~cr~ add~d to ctamplo.s 20 alon~ hitl- th~ ~ddition of th~ oaptur~ prob~ and th~ bio tin~lated riboprob~ ~both at 0.5 u~/ml) ~n~ incub~ted ~t 3 degrees for ~0 minutes. Th~ skiclcs were washed, incuba~ed wikh a SA-~P conjuj~ate, washed again, and then the si~nal ;as ¦ detected with pNPP. The second format used h~as the standard 2~ protocol with the capture probe immobilized on the dipsticl;s, the use of a TEA Cl wash step, and the addition of the liubeled probe after the capture and TEA Cl steps. The capture step was for 60 min. and the riboprobe incubation for 30 min. A ~olume of 0.5 ml was used for all rea~ents e~;cept 30l the lM ~aCl wash buffer (washes done in bulli). Results are the a~erage ~of duplicates.

Equiva1ents Those skilled in the art will recognize, or be able to ascertain by no more than routine 35 experimentation, many equivalents to tbe specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims,

Claims (56)

1. A method of isolating or determining a target nucleic acid in a sample comprising contacting the sample with a solid support coated with a substratum hybridized to a capture probe which is complementary to the target nucleic acid, under conditions appropriate for the target nucleic acid to hybridize with the capture probe.

2. A method of Claim 1, wherein the solid support comprises polystyrene.

3. A method of Claim 2, wherein the solid support is selected from the group consisting of:
polystyrene microtiter wells, polystyrene tubes and polystyrene dipsticks.

4. A method of Claim 1, wherein the substratum comprises a polynucleotide.

5. A method of Claim 4, wherein the substratum is poly(dT) or poly(dA).

6. A method of Claim 1, wherein the prehybridized oligonucleotide probe comprises an oligo-nucleotide linked to a homopolynucleotide tail section which tail section is complementary to the substratum.

7. A method of Claim 6, wherein the substratum comprises poly(dT) and the tail section comprises poly(dA).

8. A method of Claim 6, wherein the substratum comprises poly(dA) and the tail section comprises poly(dT).

9. A method of Claim 1, wherein said tail is attached to the 3' end or 5' end, or is bound to at least one internal residue of the oligonucleotide probe.

10. A method of Claim 1, wherein said oligonucleotide probe is DNA and said tail is DNA or RNA.

11. A method of Claim 1, wherein said oligonucleotide probe is RNA and said tail is DNA or RNA.

12. A method of Claim 1, wherein said tail is a homopolynucleotide sequence or a mixed base sequence of two or more nucleotides.

13. A method of Claim 1, wherein the substratum is multilayered.

14. A method of determining a target nucleic acid in a specimen comprising the steps of:

a. contacting the specimen with an agent that disrupts the molecular structure;

b. contacting the mixture obtained in (a) with a probe comprising a solid support having coated thereon a substratum to which an oligonucleotide complementary to the target nucleic acid is prehybridized, under conditions appropriate for the target nucleic acid to hybridize with the oligonucleotide probe;
c. labeling the hybridized target nucleic acids; and d. detecting the labeled targets.

15. A method of Claim 14, wherein the solid support is comprised of polystyrene.

16. A method of Claim 15, wherein the solid support comprises polystyrene microtiter wells polystyrene tubes or polystyrene dipsticks.

17. A method of Claim 14, wherein the substratum comprises a polynucleotide.

18. A method of Claim 17, wherein the substratum comprises poly(dT) or poly(dA).

19. A method of Claim 14, wherein the prehybridized oligonucleotide probe comprises an oligonucleotide linked to a polynucleotide tail section which tail section is complementary to the substratum.

20. A method of Claim 19, wherein the substratum comprises poly(dT) and the tail section comprises poly(dA).

21. A method of Claim 19, wherein the substratum comprises poly(dA) and the tail section comprises poly(dT).

22. A method of Claim 14, wherein the method further comprises washing the probe after step 6 with a tetraalkylammonium salt.

23. A method of Claim 22, wherein the tetraalkylammonium salt is selected from the group consisting of: tetraethylammonium chloride, tetraethylammonium bromide tetramethylammonium chloride and tetramethylammonium bromide.

24. A method of Claim 14, wherein the nucleic acid comprises nucleic acids derived from bacteria or viruses.

25. A method of reducing nonspecific binding of a labeled probe to the support having a target nucleic acid bound thereto, and of increasing the size of the signal, the method comprising washing said solid supports with a wash buffer appropriate to lower nonspecific binding wherein said buffer is applied after the capture of a target nucleic acid but before the addition of the labeled probe.

26. The method Claim 23 comprising washing solid supports to which target molecules are bound by means of one of the following affinity pairs:
oligo(dT)-poly(dA), oligo(dA)-poly(dT), oligo(dT)-oligo(dA), or poly(dT)-poly(dA).

27. The method of Claim 26 comprising employing a set of washing conditions in which said affinity pair is much stronger than the hybrid pairs involved in a mixed base sequence, such that nonspecific hybridization of the capture probe, or labeled probe to a pseudo-target can be substantially reduced without causing elution of the target nucleic acid from the solid support.

28. The method of Claim 27, wherein said wash buffer is a tetraalkylammonium salt.

29. The method of Claim 27, wherein said washing conditions refers to the use of a wash buffer that is or contains a tetraalkylammonium salt at a temperature between approximately one and five degrees below the dissociation temperature of the probe that possesses the lowest dissociation temperature with the target.

30. The method of Claim 27, wherein the signal is increased due to enhanced binding or enhanced hybridization of the labeled probe to the target.

31. A method of removing the effect of potential interfering substances from a sample prior to the addition of a labeled probe to the sample, comprising the steps of:
a. solubilizing the nucleic acids of each sample with a chaotropic solvent;
b. contacting the sample with solid supports to which oligonucleotides specific for the target nucleic acids are pre-immobilized thereby capturing the target nucleic acids;
c. labeling the captured target nucleic acids with a labeled probe; and d. detecting the labeled target nucleic acids.

32. The method of Claim 31 in which said interfering substances that are removed in step (b) are molecules selected from the group consisting of: endogenous biotin, endogenous avidin, endogenous biotin-binding activities, nucleic acid binding molecules, molecules that can bind the labeled probe, proteases and nucleases.

33. A method of determining the presence or amount of target nucleic acid in a sample using a reversible target capture protocol comprising the steps of:
a. providing a first capture probe comprising an oligonucleotide preimmobilized to a first substratum through a short tail;
b. contacting the probe with the sample under hybridizing conditions appropriate to allow hybridization of the target nucleic acid with the bound oligonucleotide and wherein the hybrid between the short tail of the first oligonucleotide probe with the first substratum is stable;
c. eluting the probe under conditions appropriate to destabilize the short tail-first substratum hybrid or the first oligonucleotide probe-target hybrid;
d. adding a second capture probe having a proimmobilized oligonucleotide sequence specific for the target nucleic acid, wherein said oligonucleotide is bound to the support through a long tail;
e. recapturing the target nucleic acid by means of the second capture probe;
f. labeling said target nucleic acid; and g. measuring said labeled target nucleic acid.

34. The method of Claim 34, wherein the short tail is in the range of approximately 10-20 nucleotides and the long tail is greater than about 30 nucleotides.

35. The method of Claim 33, wherein the short tail-substratum hybrid is stabilized by adding a solvent appropriate to strengthen the A-T
(A-U) base pair relative to the G-C base pair, or to strengthen the poly(dA or rA)-oligo(dT or U) or poly(dT or U)-oligo(dA or rA) or poly(dA
or rA)-poly(dT or U) homopolymer relative to mixed base sequences.

36. The method of Claim 35, wherein said solvent comprises a tetraalkylammonium salt.

37. The method of Claim 33, wherein said elution is performed by contacting the probe with a chaotropic solvent.

38. The method of Claim 38, wherein the chaotropic solvent is GuSON.

39. The method of Claim 34, wherein the second capture probe has the same mixed based sequence as the first capture probe.

40. The method of Claim 34, wherein the second capture probe has a different mixed base sequence from the first capture probe, thereby enhancing the specificity of the hybridization reaction and the assay.

41. A method of reversible target capture capable of enhancing the overail specificity of an assay, comprising the steps of:

a. providing a first solid support having a first tailed oligonucleotide probe hybridized thereto;
b. contacting a second tailed oligonucleotide probe with a second solid support, said second probe having a different mixed base sequence from said first oligonucleotide probe;
c. contacting the first solid support of (a) with a sample containing target nucleic acids, thereby capturing target nucleic acids on said first solid support;
d. eluting target nucleic acids from the first solid support;
e. contacting the target nucleic acids from (d) the second solid support thereby capturing the target nucleic acids on said second solid support;
f. labeling said target nucleic acids; and g. detecting said target nucleic acids.

42. The method of Claim 41, wherein said first solid support has a short or a long complementary nucleotide sequence attached to it.

43. The method of Claim 41, wherein elution of the target from the first solid support also removes the tailed oligonucleotide probe, said first tailed probe being prevented from binding to the second solid support because said second solid support remains substantially saturated with said second capture probe during the entire second capture process.

44. The method of Claim 41, wherein a homopolymer complementary to the tail on the first capture probe is added prior to the addition to the sample of the second solid support; said homopolymer being added in a quantity in excess over the amount required to hybridize to all of the tails on said first capture probe, and said homopolymer being incubated with said tail on the first capture probe for an appropriate length of time such that essentially no free tail is available to bind to said second solid support.

45. The method of Claim 41, wherein a homopolymer complementary to the homopolymer sequence on the second solid support is incubated with the second support for a sufficient period of time prior to the addition of the sample to said second support to prebind any free sequences on said second support, such that said first capture probe is substantially prevented from binding to said second solid support.

46. The method of Claim 41, wherein elution of the target from the first solid support does not remove said first tailed capture probe from said first solid phase because the elution occurs under a set of conditions that stabilizes the substratum-tail hybrid relative to the mixed base sequence hybrid.

47. The method of Claim 46, wherein said conditions comprise the use of a tetraalkylammonium salt for said first elution at a temperature in which the mixed base sequence hybrid is unstable while the tail-substratum hybrid is stable.

48. A kit comprising:
a. suitable solid supports, each containing a bound substratum and a specific tailed capture probe prehybridized to the substratum; and b. an agent that disrupts molecular structures .

49. A kit of Claim 48, wherein the solid supports comprise agarose beads, polystyrene microtiter wells, polystyrene tubes or polystyrene dipsticks.

50. A kit of Claim 48, wherein the substratum comprises a polynucleotide.

51. A kit of Claim 48, wherein the substratum comprises poly(dT) and the capture probe tail comprises poly(dA).

52. A kit of Claim 48, wherein the substratum comprises poly(dA) and the capture probe tail comprises poly(dT).

53. A kit of Claim 48, further comprising a wash buffer, a labeled probe, a means for detecting the labeled probe, one or more positive control samples, one or more negative control samples, elution buffers for reversible target capture, or amplification or cloning reagents.

54. A kit comprising:
a. suitable solid supports, each having a bound substratum;
b. a separate tailed capture probe, wherein the tail is complementary to the substratum; and c. an agent that disrupta molecular structures.

55. A kit comprising:
a. suitable solid supports, each containing a bound substratum and a specific capture probe prehybridized to the substratum;
b. lysis solution;
c. labeled probe;
d. wash buffer;
e. a means for detecting said labeled probe f. one or more positive control samples;
g. one or more negative. control samples;
h. elution buffers for reversible target capture; and i. amplification or cloning reagents.

56. A device comprising:
a. a pipetting station and a detection apparatus, said pipetting station being capable of performing sequential operations of adding and removing reagents to the solid phases at specified time points in a thermostatted environment;
b. said sequential operations comprising:
i. mixing samples, lysis solutions, and solid supports;
ii. withdrawing fluid;
iii. adding wash buffer;
iv. repeating steps two and three several times;

v. adding optional stringency wash buffer, or elution buffer;
vi. removing optional stringency wash buffer, or elution buffer;
vii. adding labeled probe(s);
viii. repeating steps two and three several times;
ix. adding detection agents;
x. repeating steps two and three several times; and xi. detecting signal with said detection apparatus.