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`I!, hereby certify that this transmittal letter and the
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`TRANSMITTAL LETTER FOR UNEXECUTED
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`Sir:
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`Page 9 of 95
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`A MULT]PLE DEPENDENT CLAIM
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`~'
`
`~
`
`[x]
`
`390.00
`A chec~ in the amount of $
`filing 1fee is transmitted herew1th.
`
`in payment of the
`
`[X]
`
`;,'
`
`The Co~issioner is hereby authorized to charge payment of
`any adqitional filing fees required under 37 C.F.R. 1.16 in
`connec~ion with the paper(s) transmitted herewith, or credit
`any ov~rpayment of same, to Deposit Account No. 06-1075. A
`duplic~te copy of this transmittal letter is transmitted
`herewi~h.
`
`[-]
`
`,,
`
`to Deposit Account No. 06-1075
`Pleaseicharge $
`in pay~ent of the filing fee. A duplicate copy of this
`transmittal letter is transmitted herewith.
`
`~.
`
`~
`
`s/9/?s-
`
`'f1CAA.f_ E. Bole.
`Mary E: i?'Bak
`Registration No. 31,215
`Attorney for Applicant(s)
`. · ecfo Fish & Neave
`875 Third Avenue
`New York, New York 10022
`-' .~el.: (212) 715-0600
`
`.......
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`1
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`2
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`Page 10 of 95
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`3 {)1) , (}{)- /0 7
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`732374
`
`APPLICATION
`
`FOR
`
`UNITED STATES LffiERS PATENT
`
`Specification.
`
`To ALL WHoM IT rJAY CoNcERN:
`ii
`BE It Known, tat we .~~!'!".~.~ .. ~.: ... !?.'F.~:!.l:':~~9:?.?.~~?.~.! ... P.9.H:.+.~ .. ~f.~.~~~M ....... and
`citizens
`KENNETH H. JOHliiSTON, BARBARA E. THALENFELD
`................................. t····················································· .. ·····································-················1
`of .. !::?:<:: .. :!.~~~.'::~IJ!:~!~~···~lt~§~~·~:t··~!_l.~ .. Y.~~.<. .. ~.""~~·~7z·""~%t~!?.~Er.;~~ residents,
`respectively, of ... ~§lf ... X!?XK, ... N .•. ¥..· ... JQ.Q.J.~.<.........
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`95 Horatto Street
`50 ast 9
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`3 .i~THODS ~~TURES EMPLOYING
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`of which the followi~g is a specification:
`
`Page 11 of 95
`
`
`
`ENZ 7 CIP
`
`..,~..,...,..
`
`TECHNICAL FIELD OF INVENTION
`
`BACKGROUND OF THE INVENTION
`
`f
`In the description,
`empiloyed:
`are
`,,
`{J
`_, A substance or substances, either
`. Analyte
`,
`t
`alone o~ in admixtures, whose presence is to be de-
`tected ~nd, if desired, quantitated. The analyte
`,,
`
`the following terms
`
`1-
`
`3
`
`,-..,_
`(
`:
`\
`'~
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`;\_
`f
`J
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`)''~
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`9lffil':'H~~::~~f:~~ I',':?.!:'£NYSE:fi.OT;g;m,pp&fpi,.,
`~
`This is a conti uation-in-part of appli-
`i.
`!,
`cants'. pend.ing United Sta es patent application,
`erial humber 461,469, fi ed January 21, 1983 .
`. , . . "I;;,!--·"'-
`: ( \f.J>)
`/1
`(
`. The present invention relates generally to
`the det~ction of genetic material by pol}nucleotide
`probes.!' More specifically, it relates to a method
`for quaptifiably detecting a targeted polynucleotide
`sequenc~ in a sample of biological and/or nonbiolog(cid:173)
`ical material employing a probe capable of generating
`a solub~e signal. The method and products disclosed
`'
`herein [n accordance with the invention are expected
`to be a~aptable for use in many laboratory, indus-
`trial, ~nd medical applications wherein quantifiable
`I
`and efficient detection of genetic material is
`.
`\
`des1redi~ :
`("\ .1
`\_A
`
`Page 12 of 95
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`-2-
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`;,
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`~~
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`I
`
`may bei: a DNA or RNA molecule of small or high molec(cid:173)
`ular w~ight, a molecular complex including those
`molecu~es, or a biological system containing nucleic
`t:
`acids, i· such as a virus, a cell, or group of cells.
`!
`Among the common analytes are nucleic acids (DNA and
`RNA) Of segments thereof, oligonucleotides, either
`I
`single+ or double-stranded, viruses, bacteria, cells
`in cul~ure, and the like. Bacteria, either whole or
`fragmeJts thereof, including both gram positive and
`.
`gram n~gative bacteria, fungi, algae, and other micro-
`organi~ms are also analytes, as well as animal (e.g.,
`mammal~'an) and plant cells and tissues.
`,,
`::
`Probe ..,-, A labelled polynucleotide or oligo-
`~ )
`nucleotl.ide sequence which is complementary to a poly-
`1
`nucleo~ide or oligonucleotide sequence of a particular
`analyt~, and which hybridizes to said analyte sequence.
`, Label -x That moiety attached to a poly-
`'"
`I ;
`nucleotiide or oligonucleotide sequence which com-
`,,
`prises 1:/'i signalling moiety capable of generating a
`signal l~or detection of the hybridized probe and
`analytei:. The label may consist only of a signalling
`[i
`moiety, i, e.g. , an enzyme attached directly to the
`sequencb. Alternatively, the label may be a combi-
`,,
`nation bf a covalently attached bridging moiety and
`I
`signall~ng moiety or mgioty or a combination of a
`non-cov~lently bound bridging moiety and signalling
`moiety ~hich gives rise to a signal which is detect-
`' ,,
`able, a~d in some cases quantifiable.
`:. Bridging Moiety,-:, That portion of a label
`which oh covalent attachment or non-covalent binding
`l'i
`to a po~.ynucleotide or oligonucleotide sequence acts
`' ~:
`as a li~k or a bridge between that sequence and a
`signall~ng moiety.
`' Signalling Moiety - That portion of a label
`which oh covalent attachmen{1Jr non-covalent binding
`to a polynucleotide or oligonucleotide sequence or
`I
`
`I'
`
`(/
`f
`
`0
`
`Page 13 of 95
`
`
`
`-3-
`to a br~dging moiety attached or bound to that
`' ~ ~
`sequenc~ provides a signal for detection of the label.
`' Signal --,_That characteristic of a label or
`:
`I_;
`signalling moiety -that permits it to be detected
`';' '
`from sequences that do not carry the label or
`'
`signalling moiety.
`n\!
`/': The analysis and detection of minute quan-
`tities &f substances in biological and non-biological
`I
`samplesihas become a routine practice in clinical,
`,,
`diagnostic and analytical laboratories. These detec(cid:173)
`"
`tion te~hniques can be divided into two major
`'
`classes~: ( 1) those based on ligand-receptor interac-
`' ~·
`tions (~.g., immunoassay-based techniques), and
`(2) tho~e based on nucleic acid hybridization (poly(cid:173)
`nucleoblde sequence-based techniques).
`' -
`Immunoassay-based techniques are charac-
`terized illy a sequence of steps comprising the non(cid:173)
`covalen~ binding of an antibody and antigen comple-
`mentary l,to it. See, for example, T. Chard,
`an Intrdiduction To Radioimmunoassay and Related
`~-···"· "'' jl'
`, ............ ------" ..
`--~-- .... - ~ .... ···--'
`-·· ---~---
`Techni<I\1~S ( 1978).
`---------. '. . ---.;.---
`' Polynucleotide sequence-based detection
`techniqtj~s are characterized by a sequence of steps
`compris~ng the non-covalent binding of a labelled
`~I
`polynucJleotide sequence or probe to a complementary
`1:
`sequence!. of the analyte under hybridization condi-
`,,
`tions_i1 accordance with th~Crick base
`pairing !of adenine (1}.) and~-.:
`·
`·
`(T), and
`guanine I'( G) andJf~e (C), and the detection of
`,:
`that hyl:lridization.
`[M. Grunstein and D. S. Hogness,
`l'i
`"Colonyl~ybridization: A Method For The Isolation Of
`,_
`Cloned qNAs That Contain A Specific Gene", Proc.
`Natl. Aqad. Sci. USA, 72, pp. 3961-65 (1975)]. Such
`-
`.
`'Y
`polynuc];eotide detection techniques can involve a
`~I
`fixed arlalyte [see, e.g., United States patent
`,,
`4,358,5~S to Falkow et al], or can involve detection
`
`~,
`
`~-
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`
`Page 14 of 95
`
`
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`1
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`-4-
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`of an anlalyte in solution [see U.K. patent applica-
`'
`tion 2,0119,408 A].
`The primary recognition event of polynu(cid:173)
`cleotide! sequence-based detection techniques is the
`non-covJlent binding of a probe to a complementary
`II
`•
`sequence~· of an analyte, brought about by a precJ.se
`i:
`molecul~~ alignment and interaction of complementary
`nucleot~des of the probe and analyte. This binding
`\:
`event is!: energetically favored by the release of
`i
`non-cova!lent bonding free energy, e.g., hydrogen
`~
`bonding,! stacking free energy and the like.
`In addition to the primary recognition
`event, iit is also necessary to detect when binding
`takes pl]ace between the labelled polynucleotide
`sequenc~· and the complementary sequence of the
`analyte J This detection is effected through a sig(cid:173)
`nalling/step or event. A signalling step or event
`'
`allows qetection in some quantitative or qualitative
`manner, ie. g. , a human or instrument detection system,
`of the d:ccurrence of the primary recognition event.
`~~
`The primary recognition event and the sig-
`nalling l!event of polynucleotide sequence based detec-
`1·
`tion teqhniques may be coupled either directly or
`;i
`indirectly, proportionately or inversely proportion-
`,,
`ately. !,Thus, in such systems as nucleic acid hybrid(cid:173)
`ization$ with sufficient quantities of radiolabeled
`probes, f'the amount of radio-activity is usually di(cid:173)
`rectly ~roportional to the amount of analyte present.
`Inversety proportional techniques include, for
`example( competitive immune-assays, wherein the
`,,
`amount bf detected signal decreases with the greater
`amount pi analyte that is present in the sample.
`i Amplification techniques are also employed
`i
`for enh~cing detection wherein the signalling event
`'
`is rela~ed to the primary recognition event in a
`ratio ~reater than 1:1. For example, the signalling
`componept of the assay may be present in a ratio of
`
`'
`
`r;
`
`/
`(/'
`
`Page 15 of 95
`
`
`
`--
`
`-5-
`
`~:
`
`('
`
`11.---
`
`10:1 to!each recognition component, thereby provid(cid:173)
`ing a 19-fold increase in sensitivity.
`' , A wide variety of signalling events may be
`employe~ to detect the occurrence of the primary
`!:
`recogni1ion event. The signalling event chosen
`'
`depends !'on the particular signal that characterizes
`the lab~l or signalling moiety of the polynucleotide
`sequenc~ employed in the primary recognition event.
`Althoug~ the label may only consist of a signalling
`moiety, I, which may be detectable, it is more usual
`for the!label to comprise a combination of a bridging
`'
`moiety ~ovalently or non-covalently bound to the
`polynucleotide sequence and a signalling moiety that
`jt
`is itseff detectable or that becomes detectable after
`furtherimodification.
`! The combination of bridging moiety and
`signalling moiety, described above, may be con-
`i
`•
`•
`structed before attachment or b~nd~ng to the
`~~
`sequenc$, or it may be,L_se~ J11y attached or
`i
`#
`j:
`bound t@ the sequence. For example, the bridging
`moiety *ay be first bound or attached to the sequence
`and the* the signalling moiety combined with that
`bridgin~ moiety.
`In addition, several bridging
`I
`moieties and/or signalling moieties may be employed
`~:
`together in any one combination of bridging moiety
`and si~alling moiety.
`: Covalent attachment of a signalling moiety
`or brid~ing moiety/signalling moiety combination to
`a seque~ce is exemplified by the chemical modification
`of the ~equence with labels comprising radioactive
`moieties, fluorescent moieties or other moieties
`!;
`that th~mselves provide signals to available detec(cid:173)
`t
`tion merns or the chemical modification of the
`sequencb with at least one combination of bridging
`I'
`moiety ~nd signalling moiety to provide that signal.
`,,
`Non-covalent binding of a signalling moiety
`or brid~ing moiety/signalling moiety to a sequence
`
`(,'
`
`'
`
`(:
`
`·-7
`(
`
`Page 16 of 95
`
`
`
`-6-
`
`~
`
`,,
`
`involv~ the non-covalent binding to the sequence of a
`i.i
`signal~ing moiety that itself can be detected by appro-
`priate l,means, i.e. , or enzyme, or the non-covalent
`binding to the sequence of a bridging moiety/signalling
`moiety ito provide a signal that may be detected by
`(i
`one of i'those means. For example, the label of the
`polynu~;Leotide sequence may be a bridging moiety
`'
`non-co~<l.lently bound to an antibody, a fluorescent
`moiety ior another moiety which is detectable by appro-
`'
`priate ~eans. Alternatively, the bridging moiety
`could ~e a lectin, to which is bound another moiety
`that isl detectable by appropriate means.
`~
`There are a wide variety of signalling
`moietie[s and bridging moieties that may be employed
`1:
`in labeils for covalent attachment or non-covalent
`bindin~, to polynucleotide sequences useful as probes
`in analWte detection systems. They include both a
`wide va~iety of radioactive and non-radioactive sig-
`1
`nallingi, moieties and a wide variety of non-radioactive
`bridginb moieties. All that is required is that the
`i'
`signall~ng moiety provide a signal that may be detected
`,,
`by apprbpriate means and that the bridging moiety,
`I
`if any,ibe characterized by the ability to attach
`[,
`covaleni):ly or to bind non-covalently to the sequence
`and alsb the ability to combine with a signalling
`moiety.!
`
`'
`
`Radioactive signalling moieties and com(cid:173)
`binatiobs of various bridging moieties and radio(cid:173)
`active ~ignalling moieties are characterized by one
`il'
`or morel radioisotopes such as 32p, 1311, 14c, 3H,
`6 0co, srNi, 6 3Ni and the like. Preferably, the iso(cid:173)
`tope e~loyed emits ~ or y radiation and has a long
`half l~fe. Detection of the radioactive signal is
`then, $st usually, accomplished by means of a radio-
`,,
`activi~ detector, such as exposure to a film.
`The disadvantages of employing a radio(cid:173)
`active isignalling moiety on a probe for use in the
`'
`
`,:1
`
`<:7
`,_.
`/!
`
`Page 17 of 95
`
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`-7-
`
`:;I
`
`1;:
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`~
`
`I
`identif~cation of analytes are well known to those
`skilled!: in the art and include the precautions and
`hazardsiinvolved in handling radioactive material,
`the shott life sp~ch material and the cor(cid:173)
`relativ~ly large~ involved in use of radio-
`,
`active materials.
`I
`; Non-radioactive signalling moieties and
`'
`combinations of bridging moieties and non-radioactive
`signalling moieties are being increasingly used both
`in rese~rch and clinical settings. Because these
`i
`signall~ng and bridging moieties do not involve
`'
`radioactivity, the techniques and labelled probes
`I
`using them are safer, cleaner, generally more stable
`~·
`when st~red, and consequently cheaper to use. Detec(cid:173)
`f·
`tion se~sitivities of the non-radioactive signalling
`moietie~ also are as high or higher than radio-label-
`ling tebhniques.
`I
`Among the presently preferred non-radio-
`active ~ignalling moieties or combinations of
`I
`bridging/signalling moieties useful as non-radio(cid:173)
`(
`active labels are those based on the biotin/avidin
`'
`bindingf system.
`[P. R. Langer et al., "Enzymatic
`Synthes~s Of Biotin-Labeled Polynucleotides: Novel
`Nucleici,Acid Affinity Probes", Proc. Natl. Acad. Sci.
`USA, 781, pp. 6633-:37 (1981); J.'Stavrianopoulos
`- -
`,:
`''1
`et al.,i "Glycosylated DNA Probes For Hybridization;-
`Dectionlof Homologous Sequences", presented at the(cid:173)
`Third Abnual Congress For Recombinant DNA Research
`
`0
`
`,
`{
`
`'i
`
`( 1983); I R. H. Singer and D. C. Ward, "Actin Gene
`
`Express~on Visualized In Chicken Muscle Tissue
`,,
`Culture!, By Using In Situ Hybridization With A
`Biotinted Nucleotide Analog", Proc. Natl. Acad.
`1)]. For a review of
`Sci. US~/\, 79, pp. 7331-35 (1982
`N
`non-ra~~oactive signalling and bridging/signalling
`system~', both biotin/avidin and otherwise, see D. C.
`Ward eti al., "Modified Nucleotides And Methods Of
`
`-~
`/
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`'--1
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`Page 18 of 95
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`~,
`
`,.
`
`'f
`
`-8-
`Prepar~ng And Using Same", European Patent applica(cid:173)
`tion N1. 63879.
`' Generally, the signalling moieties employed
`'
`in both radioactive and non-radioactive detection
`techniJues involve the use of complex methods for
`determ~ning the signalling event, and/or supply only
`~
`an un~antitable positive or negative response. For
`I
`exampl~, radioactive isotopes must be read by a radio-
`activi~ counter; while signalling moieties forming
`i
`insolubl1e "signals", i.e., precipitates, certain
`fluoreslcers, and the like [see, e.g., David et al.,
`I
`United ~tates Patent No. 4,376,100] only provide
`detectibn not quantitation of the analyte present in
`I:
`the tesited sample.
`' One step toward facilitating rapid and effi(cid:173)
`cient gpantitation as well as detection of the
`'
`hybridifation event was the work of Heller et al. in
`Europeap Patent Applications No. 70685 and 70687
`which ~~scribe the use of a signalling moiety which
`produce~ a soluble signal for measurable detection
`by a spbctrophotometer. These European patent appli(cid:173)
`~~
`cations!' disclose the use of two different probes com-
`plemen~ry to different portions of a gene sequence,
`II
`with e~~h probe being labelled at the end which will
`'
`.
`abut t~~ other probe upon hybridization. The first
`probe iis labelled with a chemiluminescent complex
`that e~ts lights of a specific wavelength. The
`second brobe is labelled with a molecule that emits
`light olf a different wavelength measurable by spectro-
`photomei;try when excited by the proximity of the first
`signallling moiety. However, this technique is per-
`~~
`formed ~n solution and can generate false positive
`resultsl in the absence of the analyte if the two
`probes ~appen to approach too closely in solution
`and re~~t with each other.
`'·
`Similarly, U.K. Patent Application 2,019,408A,
`publis~ed October 31, 1979, discloses a method for
`
`I
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`
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`
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`
`Page 19 of 95
`
`
`
`·)
`
`a._.,
`
`{(/
`
`-9-
`
`detecting nucleic acid sequences in solution by employ-
`'
`ing an ~nzyme-labelled RNA or DNA probe which, upon
`'
`contacti'with a chromogen substrate, provides an
`t:
`opticalfY readable signal. The analytes may be sepa-
`rated £tom contaminants prior to hybridization with
`the pro~e, or, alternatively, the hybrid probe-analyte
`may be femoved from solution by conventional means,
`i.e., c~ntrifugation, molecular weight exclusion,
`and the ilike. Like Heller's technique, this method
`is perfJrmed in solution.
`i There remains therefore a need in the art
`a r~liable, simple and quantifiable technique for
`det~ction of analytes of interest in biological
`non-fbiological samples.
`i
`
`1:'
`
`SUMMARY OF THE INVENTION
`
`for
`the
`and
`-~'"'·J
`I L\
`!
`("
`
`~
`
`f.,
`
`. The pre~ent-invention provides a solution
`.
`for the !'disadvantages of presently available methods
`of dete~ting analytes by a novel combination of
`i
`hybridi~iation and immunological techniques.
`In
`acgoraarlee with the ~factice ~ the present inven(cid:173)
`tion, c~emically labelled polynucleotide or oligo(cid:173)
`nucleot~de probes are employed to detect analytes by
`having ~e capacity to generate a reliable, easily
`quantifiable soluble signal.
`Analytes to be detected by the detection
`~
`process~s of this invention may be present in any
`biologi4al or non-biological sample, such as clinical
`samplesj for example, blood urine, feces, saliva,
`pus, se~en, serum, other tissue samples, fermentation
`broths, !culture media, and the like. If necessary,
`I
`the ana~yte may be pre-extracted or purified by known
`methods i: to concentrate its nucleic acids. Such nucleic
`I
`acid co~centration procedures include, for example,
`phenol ~xtraction, treatment with chloroform-isoamyl
`alcoholior chloroform-octanol, column chromatography
`
`I I
`
`Page 20 of 95
`
`
`
`u__..
`
`0'
`
`CL.---
`0
`
`~
`4..---
`
`~
`
`~
`
`-10-
`
`( e.g. , isephadex, hydroxyl apatite), and CsCl equi(cid:173)
`libriunl: centrifugation. The analyte, separated from
`contamibating materials, if presentJis~ according to
`!
`~
`the prefent invention, fixed in hybridizable form to
`a solidi. supp~
`I I~$(anee 1-Titl.:l. the praet:ice of ehis_
`k..
`t~
`ia.veftti~R, an&lytes in a biological sample are pre-
`'
`ferablyidenatured into single-stranded form, and
`then ditectly fixed to a suitable solid support.
`'
`Alternatively, the analyte may be directly fixed to
`,,
`the support in double-stranded form, and then dena•
`tured. !:,The present invention also encompasses
`indireci fixation of the analyte, such as in in situ
`- - -
`,.
`techni~es where the cell is fixed to the support and
`sandwic~ hybridization techniques where the analyte
`I'
`is hybridized to a polynucleo~e sequence that is
`fixed tq the solid support. J.Ja. 'Ehe praet:iees ef
`"Gl:l.is in~entio!i., i1; is preferred that the solid support
`to whic~ the analyte is fixed be non-porous and trans(cid:173)
`parent, lsuch as glass, or alternatively, plastic,
`I
`II
`polysty~ene, polyethylene, dextran, polypropylene
`I
`and the~. ike. Conventional porous materials, e.g.,
`nitroce~~ulose filters, although less desirable for
`practicel of the method of the present invention, may
`also be bmployed as a support.
`It is also highly desirable that the analyte
`be easil~ fixed to the solid support. The capability
`I•
`to easilr fix the analyte to a transparent substrate
`would permit rapid testing of numerous samples by
`the dete~tion techniques described herein.
`: Chemically-labeled probes acceniin~ te the
`iw·8~tie~ are then brought into contact with the
`'
`fixed sifgle-stranded analytes under hybridizing
`condi tiops. The probe .,ccox:aiag too Ure present
`fi
`ia.v:eatie~ is characterized by having covalently
`'
`.
`attachedlto it a chemical label which consists of a
`signallifg moiety capable of generating a soluble
`('
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`j /
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`Page 21 of 95
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`signal. Desirably, the polynucleotide or oligonucleo(cid:173)
`tide pr6pe provides sufficient number of nucleotides
`I
`in its srquence, e.g., at least about 25, to allow
`s