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`CONTENTS
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`Staple lssue SEE; Here
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`Page 6 of 95
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`INTERFERENCE SEARCHED
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`
`Page 7 of 95
`Page 7 of 95
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`

`
`
`
` PATENT APPLICATION SERIAL N0. 6
`
`U.S. DEPARTMENT OF COMMERCE
`PATENT AND TRADEMARK OFFICE
`EEE RECORD SHEET
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`8 20579 93/01/39 3359355
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`35-1135
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`191
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`Page 8 of 95
`Page 8 of 95
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`ied pro 1082
`other Than A Small Entity
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`732374
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`attorney Docket No.
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`Enz—7 CIP
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`App1iCaflt(s§= Jannis G. Stavrianopoulos et al.
`‘THVJ
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`EXPRESS MAIL CERTIFICATION
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`"fixpress Mail" mailing label number
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`3 32913141
`
`Date of deposit May 91
`
`, 19a§_.
`
`Ighereby certify that this transmittal letter and the
`other papers and fees identified in this transmittal letter as
`being transmitted herewith are being deposited with the United
`States Postal Service "Express Mail Post Office to Addressee"
`service under 37 C.F.R. l.l0 on the date indicated above and are
`addressed to the Commissioner of Patents and Trademarks,
`Washington,@D.C. 20231.
`F:
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`59(1)/wag.’
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`Hon. Commisiioner of Patents
`and Trademarks
`Washington,yD.C. 20231
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`TRANSMITTAL LETTER FOR UNEXECUTED
`ORIGINAL PATENT APPLICATION
`
`Sir:
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`firansmitted herewith for filing are the [Z1 specifi-
`[YE-claims;
`[Y1 abstract;
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`Informal drawings. Eormal drawings will be filed
`during the pendency at this application.
`
`Page 9 of 95
`Page 9 of 95
`
`’
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`

`
`The filing flée has been calculated as shown below:
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`FOR
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`NUMBER
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`NUMBER
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`A checfi in the amount of $
`filinggfee is transmitted herewith.
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`The Commissioner is hereby authorized to charge payment of
`any additional filing fees required under 37 C.F.R. 1.16 in
`connecfiion with the paper(s) transmitted herewith, or credit
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`to Deposit Account No. 06-1075.
`A
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`herewith.
`
`to Deposit Account No. 06-1075
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`A duplicate copy of this
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`Page 10 of 95
`Page 10 of 95
`
`

`
`
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`APPLICATION
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`UNITED STATES LETTERS PATENT
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`Specificafion.
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`To ALL WHOM I1‘ 1\/EA? CONCERN:
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`Page 11 of 95
`Page 11 of 95"
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`ENZ 7 (ZIP
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`5‘ ti
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`TECHNICAL FIELD OF INVENTION
`ffi
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`The present invention relates generally to
`the det%ction of genetic material by pol§nucleotide
`probes.§ More specifically, it relates to a method
`for quahtifiably detecting a targeted polynucleotide
`sequenc% in a sample of biological and/or nonbiolog-
`ical material employing a probe capable of generating
`a solubfie signal.
`The method and products disclosed
`herein in accordance with the invention are expected
`to be afiaptable for use in many laboratory,
`indus-
`trial, %nd medical applications wherein quantifiable
`and effficient detection of genetic material is
`desired%
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`BACKGROUND OF THE INVENTION
`
`In the description,
`
`the following terms
`
`f/
`I
`are employed:
`i
`E Analgte 51A substance or substances, either
`E1
`.
`.
`J
`.
`alone or in admixtures, whose presence 1s to be de-
`tected End, if desired, quantitated.
`The analyte
`
`Page 12 of 95
`Page 12 of 95
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`may beia DNA or RNA molecule of small or high molec-
`ular wbight, a molecular complex including those
`molecuies, or a biological system containing nucleic
`acids,§such as a virus, a cell, or group of cells.
`Among the common analytes are nucleic acids (DNA and
`RNA) 0% segments thereof, oligonucleotides, either
`single? or double-stranded, viruses, bacteria, cells
`in culiure, and the like. Bacteria, either whole or
`fragmeéts thereof,
`including both gram positive and
`gram négative bacteria, fungi, algae, and other micro-
`organifims are also analytes, as well as animal (e.g.,
`mammalian) and plant cells and tissues.
`:4
`Probelz A labelled polynucleotide or oligo-
`nucleo%ide sequence which is complementary to a poly-
`nucleofifide or oligonucleotide sequence of a particular
`analytéiand which hybridizes to said analyte sequence.
`v,‘
`%‘
`Label ;§That moiety attached to a polye
`nucleofiide or oligonucleotide sequence which com-
`prises % signalling moiety capable of generating a
`signalgfor detection of the hybridized probe and
`analyteg
`The label may consist only of a signalling
`moiety,%e.g., an enzyme attached directly to the
`sequenc%._ Alternatively,
`the label may be a combi-
`nation %f a covalently attached bridging moiety and
`signallfing moiety er—moiet§ or a combination of a
`non-covfilently bound bridging moiety and signalling
`moiety Qhich gives rise to a signal which is detect-
`able, ahd in some cases quantifiable.
`fi Bridging MoietyrE)That portion of a label
`which on covalent attachment or non—covalent binding
`to a poaynucleotide or oligonucleotide sequence acts
`as a li%k or a bridge between that sequence and a
`signalling moiety.
`T
`§;g§§lliEg_figi§Ey’- That portion of a label
`which oh covalent attachment or non—covalent binding
`to a poiynucleotide or oligonucleotide sequence or
`
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`Page 13 of 95
`Page 13 of 95
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`to a bridging moiety attached or bound to that
`sequenc% provides a signal for detection of the label.
`L
`Signal FsThat characteristic of a label or
`signalling moiety that permits it to be detected
`from seéuences that do not carry the label or
`signalling moiety.
`F?
`The analysis and detection of minute quan-
`tities éf substances in biological and non-biological
`samplesghas become a routine practice in clinical,
`diagnostic and analytical laboratories. These detec-
`tion teihniques can be divided into two major
`classesi (1) those based on ligand-receptor interac-
`tions (é.g.,
`immunoassay-based techniques), and
`(2) thoée based on nucleic acid hybridization (poly-
`nucleotide sequence-based techniques).
`M
`Immunoassay-based techniques are charac-
`terizediby a sequence of steps comprising the non-
`covalenifbinding of an antibody and antigen comple-
`mentaryéto it.
`See, for example, T. Chard,
`An Intrdfluction To Radioimmunoassa And Related x __~,» -
`Technigfifes (i'ié’'%'2§"")7'".'
`”' "M"
`>'
`;: Polynucleotide sequence—based detection
`techniqdes are characterized by a sequence of steps
`comprisihg the non—covalent binding of a labelled
`polynucheotide sequence or probe to a complementary
`sequencagof the analyte under hybridization condi-
`tions in accordance with the Watson-Crick base
`pairing adenine (A) andL (T), and
`guaninegxs) andiggtiéine (C), and the detection of
`that hyqiidization.
`[M. Grunstein and D. S. Eogness,
`”ColonyiHybridization: A Method For The Isolation Of
`
`Cloned §NAs That Contain A Specific Gene", Froc.
`Such
`Natl. Adad. Sci. USA, 72, pp. 396l;§5 (l975)].
`polynucheotide detection techniques can involve a
`fixed afialyte [see, e.g., United States patent
`4,358,5§5 to Falkow et al], or can involve detection
`
`vs.
`
`Page 14 of 95
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`of an afialyte in solution [see U.K. patent applica-
`tion 2,o§19,4o8 A].
`; The primary recognition event of polynu-
`cleotid%=sequence-based detection techniques is the
`non-covafient binding of a probe to a complementary
`sequencéiof an analyte, brought about by a precise
`moleculfir alignment and interaction of complementary
`nucleotfifies of the probe and analyte. This binding
`event iséenergetically favored by the release of
`non-covilent bonding free energy, e.g., hydrogen
`bondingfifistacking free energy and the like.
`5
`In addition to the primary recognition
`fit is also necessary to detect when binding
`event,
`takes piféce between the labelled polynucleotide
`sequenciiand the complementary sequence of the
`analyteii This detection is effected through a sig-
`nallingéstep or event.
`A signalling step or event
`allows detection in some quantitative or qualitative
`manner,§e.g., a human or instrument detection system,
`of the dccurrence of the primary recognition event.
`9 The primary recognition event and the sig-
`nallingfievent of polynucleotide sequence based detec-
`tion teihniques may be coupled either directly or
`indirecfily, proportionately or inversely proportion-
`ately.
`gl;‘I‘hus,
`in such systems as nucleic acid hybrid-
`ization% with sufficient quantities of radiolabeled
`probes,?the amount of radio-activity is usually di-
`rectly éroportional to the amount of analyte present.
`Inverseiy proportional techniques include, for
`example; competitive immuno-assays, wherein the
`amount pf detected signal decreases with the greater
`amount gf analyte that is present in the sample.
`? Amplification techniques are also employed
`for enh%ncing detection wherein the signalling event
`is relaied to the pfimary recognition event in a
`ratio gieater than 1:1. For example,
`the signalling
`componeht of the assay may be present in a ratiu of
`
`;;
`.
`
`Page 15 of 95
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`
`-5-
`thereby provid-
`lO:l toieach recognition component,
`ing a 1%-fold increase in sensitivity.
`i
`A wide variety of signalling events may be
`employed to detect the occurrence of the primary
`recognition event.
`The signalling event chosen
`dependsgon the particular signal that characterizes
`the label or signalling moiety of the polynucleotide
`sequence employed in the primary recognition event.
`Although the label may only consist of a signalling
`moiety,$Which may be detectable, it is more usual
`for theglabel to comprise a combination of a bridging
`moiety éovalently or non-covalently bound to the
`polynucieotide sequence and a signalling moiety that
`is itseif detectable or that becomes detectable after
`furthergmodification.
`Q The combination of bridging moiety and
`signalling moiety, described above, may be con-
`structed before attachmegt or binding to the
`sequence, or it may bekk
`attached or
`bound to the sequence.
`the bridging
`For example,
`moiety may be first bound or attached to the sequence
`and then the signalling moiety combined with that
`bridging moiety.
`In addition, several bridging
`moietieé and/or signalling moieties may be employed
`togethei in any one combination of bridging moiety
`and signalling moiety.
`; Covalent attachment of a signalling moiety
`or bridging moiety/signalling moiety combination to
`a seque%ce is exemplified by the chemical modification
`of the gequence with labels comprising radioactive
`moietieé, fluorescent moieties or other moieties
`that thbmselves provide signals to available detec-
`tion mebns or the chemical modification of the
`sequenck with at least one combination of bridging
`M moiety bnd signalling moiety to provide_that signal.
`: Non-covalent binding of a signalling moiety
`or brid§ing moiety/signalling moiety to a sequence
`
`a¥/
`
`Page 16 of 95
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`f
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`involve the non-covalent binding to the sequence of a
`signalling moiety that itself can be detected by appro-
`priategmeans, i.e., or enzyme, or the non-covalent
`binding to the sequence of a bridging moiety/signalling
`moietygto provide a signal that may be detected by
`one oféihose means.
`For example,
`the label of the
`polynuéleotide sequence may be a bridging moiety
`non-cojalently bound to an antibody, a fluorescent
`moietyior another moiety which is detectable by appro-
`priate fieans. Alternatively,
`the bridging moiety
`could as a lectin,
`to which is bound another moiety
`that imldetectable by appropriate means.
`i
`There are a wide variety of signalling
`moietie% and bridging moieties that may be employed
`in labefls for covalent attachment or non—covalent
`bindinfifito polynucleotide sequences useful as probes
`in analyte detection systems. They include both a
`wide vaiiety of radioactive and non-radioactive sig-
`nallinggmoieties and a wide variety of non—radioactive
`bridging moieties. All that is required is that the
`signallfing moiety provide a signal that may be detected
`by apprbpriate means and that the bridging moiety,
`if any,Ebe characterized by the ability to attach
`covalently or to bind non-covalently to the sequence
`and als% the ability to combine with a signalling
`moietyi
`i Radioactive signalling moieties and com-
`binatiobs of various bridging moieties and radio-
`active %ignalling moieties are characterized by one
`or moreiradioisotopes such as 32P, 1311, 14C, 3H,
`6°Co, 5%Ni, 63Ni and the like. Preferably,
`the iso-
`tope employed emits 5 or y radiation and has a long
`half lfife- Detection of the radioactive signal is
`then, fleet usually, accomplished by means of a radio-
`activifiy detector, such as exposure to a film.
`L
`The disadvantages of employing a radio-
`activegsignalling moiety on a probe for use in the
`f
`
`Page 17 of 95
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`gfiefs
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`

`
`-7- a
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`identification of analytes are well known to those
`skilledgin the art and include the precautions and
`hazardsginvolved in handling radioactive material,
`the shoit life span of s ch material and the cor-
`relativ%1y largexégéfigggée involved in use of radio-
`
`active materials.
`E 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
`signalling and bridging moieties do not involve
`radioactivity,
`the techniques and labelled probes
`using them are safer, cleaner, generally more stable
`when st§red, and consequently cheaper to use. Detec-
`tion sehsitivities of the non-radioactive signalling
`moieties also are as high or higher than radio-1abel-
`ling teghniques.
`i
`Among the presently preferred non-radio-
`active signalling moieties or combinations of
`bridginé/signalling moieties useful as non—radio—
`active labels are those based on the biotin/avidin
`bindingisystem.
`[P. R. Langer et al., "Enzymatic
`Synthesis Of Biotin—Labeled Polynucleotides: Novel
`
`NucleiciAcid Affinity Probes", Proc. Natl. Acad. Sci.
`ggg,
`78% pp. 6633137 (1981); J.‘Stavrianopoulos
`et al.,%”Glycosylated DNA Probes For Hybridization/1_
`Dectiongof Homologous Sequences", presented at the
`Third Annual Congress For Recombinant DNA Research
`(l983);%R. H. Singer and D. C. Ward, "Actin Gene
`Expression Visualized In Chicken Muscle Tissue
`Culture§By Using in Situ Hybridization With A
`Biotinafed Nucleotide Analog", froc. Natl. Acad.
`Sci. Ush, 79, pp. 7331-35 (l982)].
`For a review of
`non-radioactive signalling and bridging/signalling
`systems; both biotin/avidin and otherwise, see D. C.
`Ward en al., "Modified Nucleotides And Methods Of
`
`4L/
`
`5
`
`fi’
`
`Page 18 of 95
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`\:_.
`
`-3-
`
`Preparing And Using Same", European Patent applica-
`tion Né. 63879.
`the signalling moieties employed
`3 Generally,
`in both radioactive and non-radioactive detection
`techniéues involve the use of complex methods for
`determfihing the signalling event, and/or supply only
`an unqiantitable positive or negative response. For
`example} radioactive isotopes must be read by a radio-
`activifiy counter; while signalling moieties forming
`insolufile "signals", i.e., precipitates, certain
`fluoresters, and the like [see, e.g., David et al.,
`United fitates Patent No. 4,376,100] only provide
`detectifn not quantitation of the analyte present in
`the tesfied sample.
`i‘
`one step toward facilitating rapid and effi-
`cient qtantitation as well as detection of the
`hybridihation event was the work of Heller et al.
`Europeah Patent Applications No. 70685 and 70587
`which dkscribe the use of a signalling moiety which
`producet a soluble signal for measurable detection
`by a spkctrophotometer. These European patent appli-
`cationsgdisclose the use of two different probes com-
`plement%ry to different portions of a gene sequence,
`with eakh probe being labelled at the end which will
`abut thp other probe upon hybridization.
`The first
`probe it labelled with a chemiluminescent complex
`that emhts lights of a specific wavelength.
`The
`second probe is labelled with a molecule that emits
`light 0% a different wavelength measurable by spectro-
`photomatry when excited by the proximity of the first
`signallhng moiety. However, this technique is per-
`formed fin solution and can generate false positive
`result& in the absence of the analyte if the two
`probes %appen‘to approach too closely in solution
`and reict with each other.
`K
`it
`similarly, U.K. Patent Application 2,019,4o8A,
`publisfied October 31, 1979, discloses a method_fori
`1
`
`in
`
`Page 19 of 95
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`E
`£2
`
`-9-
`
`detectiig nucleic acid sequences in solution by employ-
`ing an enzyme—labelled RNA or DNA probe which, upon
`contactgwith a chromogen substrate, provides an
`°PtiCa1%Y readable signal.
`The analytes may be sepa*
`rated from contaminants prior to hybridization with
`the pro%e. or. alternatively,
`the hybrid probe-analyte
`may be removed from solution by conventional means,
`i-E-. centrifugation, molecular weight exclusion,
`and theilike. Like Hel1er‘s technique, this method
`is perfdrmed in solution.
`There remains therefore a need in the art
`
`for a reliable, simple and quantifiable technique for
`the detection of analytes of interest in biological
`L;
`_
`.
`and nonjbiological samples.
`T
`
`SUMARY OF THE INVENTION
`
`/-—..
`.3.)
`
`i The present—invention provides a solution
`for theidisadvantages of presently available methods
`of deteiting analytes by a novel combination of
`hybridiiation and immunological techniques.
`In
`acee£ée€ee—with—Hm1n%miice—e£
`the present inven-
`tion, chemically labelled polynucleotide or oligo—
`nucleotide probes are employed to detect analytes by
`having fihe capacity to generate a reliable, easily
`quantifiable soluble signal.
`Analytes to be detected by the detection
`
`:‘_-I’;
`
`I
`rz
`
`J
`
`61/,
`
`4L/
`
`processes of this invention may be present in any
`biologiqal or non—biological sample, such as clinical
`samplesé for example, blood urine, feces, saliva,
`pus, sefien, serum, other tissue samples, fermentation
`broths,:culture media, and the like.
`If necessary,
`the anaiyte may be pre-extracted or purified by known
`methodsito concentrate its nucleic acids.
`Such nucleic
`acid coicentration-procedures include, for example,
`phenol éxtraction,
`treatment with chloroform—isoamyl
`alcoholior chloroform—octano1, column chromatography
`
`;
`
`Page 20 of 95
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`

`
`-10..
`
`(e.g.,%Sephadex, hydroxyl apatite), and Cscl equi-
`libriufiicentrifugation.
`The analyte, separated from
`contami%ating materials, if presentkisx according to
`the prefent invention, fixed in hybridizable form to
`a solidgsuppzrtg E
`
`k
`I
`iaventi9nT—analytes-in a biological sample are pres
`ferablygdenatured into single-stranded form, and
`then diiectly 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
`indirect fixation of the analyte, such as in in situ
`techni
`fies where the cell is fixed to the support and
`
`i;r
`
`sandwich hybridization techniques where the analyte
`is hybridized to a polynucleoti e sequence that is
`.
`L‘
`.
`n
`,
`.
`Ln—the—praet1ees—e#
`tired to the solid support
`A”
`thss—tn*ent:on,—it is preferred that the solid support
`to whicfl_the analyte is fixed be non—porous and trans-
`parent,;such as glass, or alternatively, plastic,
`polystynene, polyethylene, dextran, polypropylene
`
`and the dike. Conventional porous materials, e.g.,
`
`nitrocehlulose filters, although less desirable for
`practicéiof the method of the present invention, may
`also be kmployed as a support.
`% It is also highly desirable that the analyte
`be easilfi fixed to the solid support.
`The capability
`to easilir fix the analyte to a transparent substrate
`would permit rapid testing of numerous samples by
`the detektion techniques described herein.
`Chemically-labeled probes wm
`.innen:io% are then brought into contact with the
`fixed si gle-stranded analytes under hybridizing
`conditiops.
`"The probe acco;ééng—to—the‘prEsent
`aEa¥eatie% is characterized by having covalently
`attachedgto it a chemical label which consists of a
`signallihg moiety capable of generating a soluble
`K
`.ru
`
`6L’
`
`4/
`
`5/
`Cc’
`
`6L,’
`Q//
`
`Page 21 of 95
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`

`
`-11-
`
`the polynucleotide or oligonucleo-
`signal.:jDesirably,
`tide pro%e provides sufficient number of nucleotides
`in its spquence, e.g., at least about 25,
`to allow
`stable hfibridization with the complementary nucleo-
`tides of?‘ the analyte.
`The hybridization of the probe
`to the sgngle-stranded analyte with the resulting
`formatioh of a double-stranded or duplex hybrid is
`then dethctable by means of the signalling moiety of
`the chemfical label which is attached to the probe
`portion pf the resulting hybrid. Generation of the
`soluble %ignal provides simple and rapid visual
`detectioh of the presence of the analyte and also
`providesia quantifiable report of the relative amount
`of analyie present, as measured by a spectrophoto-
`meter orithe like.
`i The method of the present invention involv-
`ing the holorimetric or photometric determination of
`the hybrfidized probes employs as the signalling moiety
`reagentsgwhich are capable of generating a soluble
`signal, %.g., a color change in a substrate in solu-
`tion. Pteferable components of the signalling moiety
`include hnzymes, chelating agents and co-enzymes,
`which an? able to generate colored or fluorescent
`soluble %ignals. Specifically, certain chromogens
`upon cofitact with certain enzymes are utilizable in
`the metfiod of the present invention.
`The following
`Table Iilists exemplary components for the signalling
`moiety if the present invention. Each chromogen
`listed is reactive with the corresponding enzyme to
`produceéa soluble signal which reports the presence
`of the éhemically—labeled probe analyte hybrid. The
`supersciipt notation (*)
`indicates that the chromogen
`fluoresées, rather than produces a color change;
`
`Page 22 of 95
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`
`

`
`-12,
`
`T A B L E
`
`I
`
`7-Us?”
`
`EEEL
`
`CHOMOGEN
`
`alkaline phosphatase
`
`0].’
`
`*4—Methylumbelliferyl
`phosphate
`
`acid phosphatase
`
`peroxidase
`
`B-D-galactosidase
`
`*bis (4—Methylumbelli—
`feryl phosphate
`3-0-methylfluorescein.
`*Flavone-3-diphosphate
`triammonium salt
`p-nitrophenyl phosphate
`2Na.
`
`*Tyramine hydro-
`chloride
`
`='=3- (p-1_1Y<1¥0xYP1_1enY1 >
`Pr0plOnlC acid
`*p-Hydroxyphenethyl
`alcohol
`2,2*-Azino—Di-3-
`Ethylbenzthiazoline
`sulfonic acid
`(ABTS)
`ortho-phenylenedia-
`mine 2HCl
`O—dianisidine
`*5-aminosalicylic acid
`p-cresol
`3,3'-dimethyloxy-
`benzidine
`
`3-methyl-2—benzo-
`thiazoline hydra-
`zone
`
`tetramethyl benzidine
`
`0-nitrophenyl fi-D-
`galactopyranoside
`4-methylumbelliferyl-
`E-D-galactoside
`
`glucose—oxidase
`
`ABTS
`
`i
`
`i
`
`As another aspect of the present invention,
`the si%nalling moiety may be attached to the probe
`throughgthe formation of a bridging entity or
`complefi. Likely candidates for such a bridging
`entitybwould include a biotin—avidin bridge, a
`E
`biotinsstreptavidin bridge, or a sugar-lectin bridge.
`
`Page 23 of 95
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`4;/
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`hi
`
`-13-
`
`_ Once the fixed probe-analyte hybrid is
`formed,§the method may further involve washing to
`separate any non-hybridized probes from the area of
`the support. The signalling moiety may also be
`attached to the probe through the bridging moiety
`after the washing step to preserve the materials
`employed. Thereafter, another washing step may be
`employei to separate free signalling moieties from
`those aétached to the probe through the bridging
`mo1ety.§.
`éfibihafiaqahw/pamw;Lk)
`; Broadly,L
`‘
`'
`‘
`p£evidei%ybridization techniques which provide the
`same benhfits as enzyme linked immunosorbent assay
`techniquhs, i.e,
`the qualitative and quantitative
`determin%tion of hybrid formation through a soluble
`signal. Evarious techniques, depending upon the
`chemicalflabel and signalling moiety of the probe,
`may be ehployed to detect the formation of the probeéb
`analyte hybrid.
`It is preferred, however, is-the
`pIaCIiL£%s4aE—this*invention*_to employ spectrophoto-
`metric techniques and/or colorimetric techniques for
`the deteimination of the hybrid. These techniques
`permit nét only a prompt visual manifestation of the
`soluble éignal generated by the signalling moiety
`on the d@uble—stranded hybrid, but also permit the
`quantitative determination thereof, i.e., by the
`enzymatié generation of a soluble signal that can be
`quantitatively measured.
`i Yet another aspect of the method of the
`present invention involves generating the soluble
`signal fiom the probe-analyte hybrid in a device
`
`capable of transmitting light therethrough for the
`detection of the signal by spectrophotometric tech-
`niques. %Examples of devices useful in the spectro-
`photometric analysis of the signal include conven-
`
`tional apparatus employed in diagnostic laboratories,
`i.e., plastic or glass wells,
`tubes, cuvettes or
`
`Page 24 of 95 ‘
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`i
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`-14-
`
`It may
`tubes or cuvettes.
`arrangements of wells,
`also be desirable for both the solid support to which
`the aéalyte is fixed and the device to be composed
`of the same material, or for the device to function
`as the support in addition to facilitating spectro-
`photometric detection.
`:
`A further aspect of the present invention
`provides products useful in the disclosed method for
`detecéion of a polynucleotide sequence. Among these
`produdts is a device containing a portion for retain-
`ing agfluid.
`Such portion contains an immobilized
`polynqcleotide sequence hybridized to a polynucleotide
`or olifionucleotide probe.
`The probe, as described
`above,Ehas covalently attached thereto a chemical
`label fincluding a signalling moiety capable of gener-
`ating % soluble signal. Also part of the device is
`a soluble signal, preferably a colored or fluorescent
`product, generatable by means of the signalling
`moietyg
`The portion of the device for containing
`the flpid is desirably a well, a tube, or a cuvette.
`A related product of the invention is an apparatus
`comprising a plurality of such devices for containing
`a fluié,
`in which at least one such device contains
`the ab%Ve—described immobilized polynucleotide
`sequen%e, polynucleotide or oligonucleotide probe,
`signalling moiety, and soluble signal. Additionally
`the present invention provides for the novel product
`of a n%n—porous solid support to which a polynucleo-
`tide is directly fixed in hybridizable form.
`Such a
`fixed éequence may be hybridized to another poly-
`nucleoiide sequence having covalently attached thereto
`a chemical label including a signalling moiety capable
`of gen%rating a soluble signal. As indicated above,
`the support is preferably transparent or translucent.
`such pioducts could be advantageously emp