111111 1111111 l 1111 ll 111111111 111 11111 1111 ll 11111! I111 I111 111
`
`US007064l97B1
`
`(12) United States Patent
`Rabbani et a1.
`
`(10) Patent N0.:
`45 Date of Patent:
`
`US 7,064,197 B1
`7
`*Jun. 20 2006
`
`(54) SYSTEM, ARRAY AND NON-POROUS SOLID
`SUPPORT COMPRISING FIXED OR
`IMMOBILIZED NUCLEIC ACIDS
`’
`
`4,166,103 A
`4,166,104 A
`4,228,237 A
`4,230,797 A
`
`S/1979 Wagner et a1. ............... .. 424.-'1
`8/1979 Wagner or :11.
`..
`.. 424/1
`
`10/1980 He-vey et al.
`.. 4'35/7
`1'0/1980 Boguslaski et al.
`.......... .. 435/7
`
`_
`‘
`111’: 19530 R191“?
`11/.1980 D‘e1.uca-Mclalroy ......... .. 435/S
`$9136
`_
`Pp“
`......... 260/326
`Eoguslaski et al.
`6/1981 fling ......................... 424/1
`7/1981 Sugiura et :11.
`.
`{424/1
`11/1981
`11111111 1 1.
`...... ..
`. 23/2303
`.1/1982 Matlitfsscein et a1.
`435,-'7
`3/1982 Boguslaski et :11.
`.......... .. 435/7
`3/1982 B1111 1:11.
`435/7
`3/.1982 Hmimty at all 0
`M 435/7
`I 1/.1982’ Imkow at al’ 0
`_ 4355
`2/1933 Litnian et a1.
`..
`.. 435/-7
`3/1983 David etal.
`.. 436/‘S13
`4/1983 Boguslaski et at
`435/7
`5/1983 I3_ogus1asl<i’etal.
`.......... .. 435/,7
`7‘/1983 Litinanet .11.
`............... .. 435.7
`5/1984 Self ............. ..
`.. 435/7
`11/1984 Gillespie et al.
`.. 435/6
`12/1984 Rankiet al.
`436/V504
`5/1985 Fusek
`5/1985 Urdea
`8/1985 Elings
`
`
`
`’,
`
`4,271:140 A
`4,280,992 A
`4,302,204 A
`4,312,944 A
`4,318,980 A
`4,318,981 A
`4318982 A
`43 58515 A
`4,374,925 A
`4,376,110 A
`4,380,580 A
`4,383,031 A
`4,391,904 A
`4,446,231
`4,483,920
`4,486,539
`4,516,833
`4,517,338
`4,537,861
`
`AAA‘
`
`AAA
`
`(Continued)
`
`FOREIGN PATENT DOCUMENTS
`
`DE
`
`P2618419
`
`4./1976
`
`(Continued)
`OTHER PUBLICATIONS
`
`Manuelidis et al. (1982) Journal of Cell Biology, vol. 95, pp.
`619-62535‘
`
`(Continued)
`
`Primary Exan1iner—----——Jol1n S. Brusca
`(74) Allorney, Agent, or Firm——R011ald C. Fedus
`
`(57)
`
`ABSTRACT
`
`Nucleic acids are fixed or imniobilized lo 11on—porous solid
`supports (substrates), and include systems containing such
`supports and arrays with fixed or ininiobilized nucleic acids.
`These coinpositions are useful for nucleic acid analyses and
`a host of applications, including, for example, detection,
`niutational analysis and quantification. The 11011-porous solid
`supports can be transparent or translucent, and the surfaces
`can be treated with agents to fix or i111111obili7,e the nucleic
`acids. Such agents include, for example, ainine providing
`coinpounds, epoxy compounds and acid solutions. '1he fixed
`or iininobilized nucleic acids ca11 be unlabeled, or labeled
`with at least one non—radioactive signaling moiety, such as
`the case when the nucleic acids are double-stranded.
`
`238 Claims, N0 Drawings
`
`BD EXHIBIT 1001
`
`41234553 A
`4,234,681 A
`2
`~
`
`~
`
`'
`
`1
`
`(75)
`
`Inventors: Elazar Rabbani, New York, NY (US);
`Jannis G. sta‘,l.ian0p0u]0s_ Bayshol-3,
`NY (US); 110111-e Kirtikar, Fresh
`Meadows, NY‘ (US); Kenneth H.
`Johnston’ New Orleans: LA (US);
`Ifigbara E’ Thaknfcld’ NewY°rk‘ NY
`(1
`)
`.
`..
`.
`,
`.
`..
`(73) Asslgneei EV“ hf" 5°'““.“s’ .I"°’ “/0 EM”
`B"’°h°"’= I“°'> I‘3m““g‘l*‘le= NY (US)
`.
`.
`.
`.
`Subjectto any d1scla1rner,_t11e term of 11118
`patent is extended or adjusted under 35
`U59 154(5) by 0 daY3-
`,
`,
`,
`V
`,
`,
`T195 PW“ ‘S SW19‘-‘T ‘O “ lemlml ms‘
`Clalmel
`
`.
`.
`,
`.
`(1 ) Notice:
`
`(21) Appl. No.: 08/486,070
`
`(22) Filed:
`
`Jun. 7, ".1995
`
`Related U.S:. Application Data
`
`(63) Continuation of application No. 07/967,646, filed on
`Oct. 28, 1992, now abandoned, which is a continua-
`tion of application No‘. 07/607,347, filed on Oct. 30,
`1990, now abandoned, which is a continuation of
`application No. 07/385,986, filed on Jul. 20, 1989,
`now Pat. No. 4,994,373, which is a continuation of
`application No. 06/732,374, filed on May 9, 1985,
`now abandoned, which is a continuation-in—part of
`application No. 06/461,469, filed on Jan. 27, 1983,
`now abandoned.
`
`(51)
`
`Int. C1.
`(2006.01)
`C07}! 21/04
`t
`-
`(2006.01)
`CIZN 16/11
`(52) U.S. Cl. ' ................................. .. 536/24.3; 536./25.32
`(58) Field of Classification Search .................. .. 435/6,
`435/287, 810, 283.1, 285.1, 287.1, 287.2,
`435/2877, 287.9, 288.7, 289.1, 297.1, 299.1;
`436/501; 536/22,]; 935/78, 77, 88; 422/50,
`422/55, 56, 57, 68.1, 69, 82.05, 82.06, 82.07,
`422/82.08
`
`See application file for complete search history.
`
`(56)
`
`References Cited
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`....................... .. 195/63
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`‘
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`
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`

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`US 7,064,197 B1
`Page 2
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`U .S. PATENT DOCUMENTS
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`
`................. .. 536./29
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`................. .. 435/6
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`4,581,333 A
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`4,732,847 A ”‘
`
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`536./27
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`English Translation for'Method in Nucleic Acid Research,
`“DNA-DNA Hybridization”, Kyoritsu Shuppan Co., Ltd.,
`1973, pp. 60-67 and 242.
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`pp. 727, 507, 450 and one other page.
`English Translation for Dictionary of Biochexnistry, Tokyo
`Kagaku Douj in, l984,pp. 727, 507, 450 and one other page.
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`A
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`in Enzymology 1001266-285
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`Page 4 of18
`
`

`
`US 7,064,197 B1
`
`1
`SYSTEM, ARRAY AND NON-POROUS SOLID
`SUPPORT COMPRISING FIXED OR
`IIVIMOBIIJIZEI) NUCLEIC ACIDS
`
`CROSS-REFERENCE TO OTHER RELATED
`APPLICATIONS
`
`This is a continuation application of US. Patent Appli-
`cation Ser. No. 07/967,646, filed on Oct. 28, 1992, now
`abandoned, which application is a continuation application
`of US. Patent Application Ser. No. 07/607,347, filed on Oct.
`30, 1990, also abandoned. Ser. No. 07/607,347 is a continu-
`ation of US. Patent Application Ser. No. 07/385,986, filed
`on Jul. 20, 1989, now U.S. Pat. No. 4,994,373 issued on Feb.
`19, 1991. Ser. No. 07/385,986 is a continuation of U.S.
`Patent Application Ser. No. 06/732,374, filed on May 9,
`1985, also abandoned, which application is a continuation-
`in-part of U.S. Patent Application Ser. No. 06/461,469, filed
`on Jan. 27, 1983, also abandoned.
`
`T ECHNICAI, FIELD OF INVENTION
`
`The present invention relates generally to the detection of
`genetic material by polyrrucleotide probes. More specifi-
`cally,
`it relates to a method for quantifiably detecting a
`targeted polynucleotidc sequence in a sample of biological
`and/or nonbiological material employing a probe capable of
`generating a ‘soluble signal. The method and products dis.-
`closed herein in accordance with the invention are expected
`to be adaptable for use in many laboratory, industrial, and
`medical applications wherein quantifiable and efiicient,
`detection of genetic material is desired.
`
`BACKGROUND OF THE lNVIiNTlO.N
`
`‘In the description, the following terms are employed:
`Analyte4A substance or substances, either alone or in
`admixtures, whose presence is to be detected and, if desired,
`quantitated. The analytc may be a DNA or RNA molecule of‘
`small or high molecular weight, a molecular complex
`including those molecules, or a biological system containing
`nucleic acids, such as a virus, a cell, or group of cells.
`Among the common analytcs are nucleic acids (DNA and
`RNA) or segments thereof, oligonucleotides, either single-
`or double-stranded, viruses, bacteria, cells in culture, and the
`like. Bacteria, either whole or fragnrents thereof, including
`both gram positive and gram negative bacteria, fungi, algae,
`and other microorganisms are also analytes, as well as
`animal (e.g., mammalian) and plant cells and tissues.
`Probe---——----A labelled polynucleotide or oligonucleotide
`sequence which is complementary to a polynucleotide or
`oligonucleotide sequence of a particular analyte and which
`hybridizes to said analyte sequence.
`Label—That moiety attached to a polynucleotide or oli-
`gonucleotide sequence which comprises a signalling moiety
`capable of generating a signal for detection of the hybridized
`probe and analyte. The label may consist only ofa signalling
`moiety, e.g., an enzyme attached directly to the sequence.
`Alternatively, the label may be a combination ofa covalently
`attached bridging moiety and signalling moiety or a com-
`bination of a non~covalently bound bridging moiety and
`signalling moiety which gives rise to a signal which is
`detectable, and in some cases quantifiable.
`Bridging Moiety------That portion of a label which on
`covalent attachment or non—covalent binding to a polynucle—
`otide or oligonucleotidc sequence acts as a ‘link or a bridge
`between that sequence and a signalling moiety.
`
`2
`Signalling Moiety» That portion of a label which on
`covalent attachment or non-covalent binding to a polynucle-
`otide or oligonucleotide sequence or to a bridging moiety
`attached or bound to that sequence provides a signal for
`detection of the label.
`’Signal—That characteristic of a label or signalling moiety
`that permits it to be detected from sequences that do not
`carry the label or signalling moiety.
`The analysis and detection of minute quantities of sub-
`stances irrbiological and non—biological samples has become
`a routine practice in clinical, diagnostic and analytical
`laboratories. These detection techniques can be divided into
`two major classes: (1) those based on ligand-receptor inter-
`actions (e.g., immunoassay-based techniques), and _(2) those
`based on nucleic
`acid hybridization (polynucleotide
`sequence-based techniques).
`Immunoassay-based techniques are characterized by a
`sequence of steps comprising the non—covalent binding of an
`antibody and antigen complernentary to it. See, for example,
`T. Chard, An Introduction To Radioimrmmoassay And
`Related Techniques (1978).
`Polyrrucleotide sequence-based detection techniques are
`clraracterized by a sequence of steps comprising the non-
`covalent binding of a‘ labelled. polynucleotide sequence or
`probe to a complementary sequence of the analyte under
`hybridization conditions in accordance with the Watson-
`Crick base" pairing of adenine (A) and thymine CT), and
`guanine (G) and cytosine (C), and the detection of that
`hybridization. [M. Grunstein and D. S. Hogness, “Colony
`I-lybridization: A Method For The Isolation Of Cloned
`DNAS That Contain A Specific Gene”, Proc. Natl. Acad. Sci.
`USA, 72, pp. 3961-65 (l975)]. Suclrpolynucleotide detec-
`tion techniques can involve a fixed analyte [see_. eg., U.S.
`Pat. No. 4,358,535 to Falkow et al], or can involve detection
`of an analyte in solution [see U.K. patent application 2,019,
`408 A].
`event of polynucleotide
`The primary recognition
`sequence-based detection techniques is the non-covalent
`binding of a probe to a complementary sequence of an
`analyte, brought about by a precise molecular alignment and
`interaction of complementary nucleotides of the probe and
`analyte. This binding event is energetically favored by the
`release of non—covalent bonding free energy, e.g., hydrogen
`bonding, stacking free energy and the like.
`is also
`In addition to the primary recognition event, it
`necessary to detect when binding takes place between the
`labelled polynucleotide sequence and the complementary
`sequence of the analyte. This detection is efiected through a
`signalling step 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 occurrence of
`the primary recognition event.
`The primary recognition event and the signalling event of
`polynucleotide sequence based detection techniques may be
`coupled either directly or indirectly, proportionately or
`inversely proportionately. Thus, in such systems as nucleic
`acid hybridi7ations with sufl"1cient quantities ofradiolabelcd
`probes,
`the amount of radio-activity is usually directly
`proportional
`to the amount of analyte present.
`lnversely
`proportional techniques include. for example. competitive
`immuno—assays, wherein the amount of detected signal
`decreases with the greater amount of analyte that is present
`in the sample.
`Amplilication techniques are also employed for enhanc-
`ing detection wherein the signalling event" is related to the
`primary recognition event in a ratio greater than 1:]. For
`example, the signalling component of the assay may be
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`L...) U:
`
`40
`
`50
`
`60
`
`65
`
`Page 5 ofl8
`
`

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`3
`
`US 7,064,197 B1
`
`5
`
`4
`material, the short life span of such material and the cor-
`relatively large expenses involved in use of radioactive
`materials.
`
`Non-radioactive signalling moieties and combinations of
`bridging moieties and non-radioactive signalling moieties
`are being increas_ingly -used both in research 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
`stored, and consequently cheaper to use. Detection sensi-
`tivities of the nonradioactive signalling moieties also are as
`high or higher than radio—labellin'g’ teclmiques.
`Among the presently preferred non-radioactive signalling
`moieties or combinations of bridgingsignalling moieties
`useful as non-radioactive labels are those based on the
`biotin/avidin binding system. H’. R. Langer ct al., “Enzy-
`matic Synthesis Qf Biotin-Labeled Polynucleotides: Novel
`Nucleic Acid Aflinity.Prob'es”, Proc. Nail. Acad. Sci. USA,
`78, pp. 36.633--'37 (1981); J. Stavrianopoulos et al., “Glyco-
`sylated. DNA Probes For Hybridization/Diection Of I1omolo-
`gous Sequences”, presented at the Third Annual Congress
`For Recombinant DNA Research (1983); R. H. Singer and
`D. C. Ward, “Actin Gene Expression Visualized 1n Chicken
`.Muscle Tissue Culture By Using In Situ Hybridization With
`A Biotinated Nucleotide Analog”, Proc. Natl. Acad. Sci.
`USA, 79, pp. 7331-35 (1982)]. For a review of non-mdio-
`active signalling ‘and bridging/signalling systems, both
`biotin/c vidinand otherwise, see D. C. Ward et al., “Modified
`Nucleotides And Methods Of Preparing‘ And Using Same”,
`liiuropean Patent application No. 63879.
`The above-referenced U.S. Patent Application Ser. No.
`06/255,223 was abandoned in favor of continuation appli-
`cation, U.S. Patent Application Ser. No. 06/496,915, filed on
`May 23, 1983, now US. Pat. No. 4,711,955. A related
`divisional application of the aforementioned Ser. No.
`06/496,915 was filed (on Dec. ‘8, 1987) as US. Patent
`Application Ser. No. 07/130,070 and issued on Jul. 12, 1994
`as U.S. Pat. No. 5,328,824. Two related. continuation appli-
`cations of the aforementioned Ser. No. 07/ 130,070 were
`filed on Feb. 26, 1992’ (as Ser. No. 07/841,910) and on May
`20, 1992 as (Ser. No. 07/886,660). The aforementioned
`applications, Ser. No. 07/886,660 and Ser. No. 07/841,910,
`issued as U.S. Pat. Nos. 5,449,767 and 5,476,928 on Sep. 12,
`1995 and Dec. 19, 1995, respectively. The above-referenced
`U.S. Patent Application Ser. No. 06/391,440, filed on Jun.
`23, 1982, was abandoned in favor ofU Patent Application
`Ser. No. 07/140,980, filed on Jan. 5, 1988, the latter now
`abandoned. Two divisional applications of the aforemen-
`tioned Ser. No. 07/140,980, U.S. Patent Applications Ser.
`Nos. 07/532,704 (filed o11 Jun. 4, 1990 for “Base Moiety
`Labeled Detectable Nucleotide”) and 07/567,039 (filed on
`Aug. 13, 1990 for ~“Saccharide Specific Binding System
`Labeled Nucleotides”) issued as U.S. Pat. Nos. 5,241,060
`(Aug. 31, 1993) and 5,260,433 (Nov. 9, 1993), respectively.
`The disclosures of the above—identified PN/1S article (P. R.
`Langer et a1., “Enzymatic Synthesis of Biotin—Labeled Poly-
`nucleotides: Novel Nucleic Acid Aillinity Probes,” Proc.
`Natl. Acad. Sci.{US4) 78:6633-6637 (1981) and US. Pat.
`Nos. 4,711,955, 5,328,824, 5,449,767, 5,476,928, 5,241,
`060, 5,260,433, and 4,358,535‘ are herein incorporated and
`made part of this disclosure.
`Generally, the signalling moieties employed in both radio-
`active and non-radioactive detection techniques involve the
`use of complex methods for determining the signalling
`event, and/or supply only an unquantitable positive or nega-
`tive response. For example, radioactive isotopes must be
`read by a radioactivity counter; while signalling moieties
`
`present in a ratio of 10:1 to each recognition component,
`thereby providing a 10-fold increase in sensitivity.
`A wide variety of signalling events may be employed to
`detect the occurrence of the primary recognition event. The
`signalling event chosen depends on the particular signal that
`characterizes the label or signalling moiety of the polynucle-
`otide 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 the label to
`comprise a combination of a bridging moiety covalently or 10
`non-covalently bound to the polynucleotide sequence and a
`signalling moiety that is itself detectable or that becomes
`detectable after further modification.
`The combination of bridging moiety and signalling moi-
`ety, described above, may be constructed before attachment
`or binding to the sequence, or it may be sequentially
`attached or bound to the sequence. For example, the bridg-
`ing moiety may be first bound or attached to the sequence
`and then the signalling moiety combined with that bridging
`moiety. lnaddition, several bridging moieties and/or signal-
`ling moieties may be employed together in any one combi-
`nation of bridging moiety and signalling moiety.
`Covalent attachment of a signalling moiety or bridging
`moiety/signalling moiety combination to a sequence is
`exemplified by the chemical modification of the sequence
`with labels comprising radioactive moieties, fluorescent
`moieties or other moieties that themselves provide signals to
`available detection means or the chemical modification of
`the sequence with at least one combination of bridging"
`moiety and signalling moiety to provide that signal.
`Non-covalent binding of a signalling moiety or bridging
`moiety/signalling moiety to a sequence involve the non-
`covalent binding to the sequence ofa signalling moiety that‘
`itself can be detected by appropriate means, i.e., or enzyme,
`or the non-covalent binding to the sequence of a bridging
`moiety/signalling moiety to provide a signal that may be
`detected by one of those means. For example, the label of the
`polynucleotide‘ sequence may be _a. bridging moiety non-
`covalently boundto an antibody,
`21 fluoresc