(12) United States Patent
`Rabbani et a].
`
`(10) Patent N0.:
`(45) Date of Patent:
`
`US 7,064,197 B1
`*Jun. 20, 2006
`
`US007064197B1
`
`8/1979 Wagner et a1. ............... .. 424/1
`4,166,103 A
`8/1979 Wagner et a1.
`.... .. 424/1
`4,166,104 A
`4,228,237 A 10/1980 Hevey et a1. ................ .. 435/7
`4,230,797 A 10/1980 Boguslaski et al. .......... .. 435/7
`4,234,563 A 11/1980 Rl-PPe
`4,234,681 A 11/1980 DeLuca-McElroy ......... .. 435/8
`4,251,514 A
`2/1981 R{PP@
`_
`4,254,097 A
`3/1981 Rlppe
`4,261,893 A
`4/1981 Boguslaskl et al. ....... .. 260/326
`i
`gizniisng
`424“
`4,280,992 A
`7/1981 Sugiura et a1.
`.... .. 424/1
`
`4,302,204 A 11/1981 Wahl et al. ......... ..
`.. 23/230.3
`4 312 944 A
`1/1982 Mattiasson et al. .......... .. 435/7
`4,318,980 A
`3/1982 Boguslaski et al.
`.... .. 435/7
`
`(54) SYSTEM, ARRAY AND NON-POROUS SOLID
`SUPPORT COMPRISING FIXED OR
`IMMOBILIZED NUCLEIC ACIDS
`
`(75) Inventors: Elazar Rabbani, New York, NY (US);
`Jannis G_ stavrianopouloss Bayshorea
`NY (US); Dollie Kirtikar, Fresh
`Meadows, NY (US); Kenneth H.
`Johnston’ New Orleans’ LA (Us);
`Barbara E. Thalenfeld, New York, NY
`(Us)
`
`.
`.
`.
`(73) Asslgnee: Elm’ Llfe sclences’ .lnc‘ c/O EH10
`Blochem’ Inc" parmmgdale’ NY (Us)
`
`( * ) Notice:
`
`
`
`Subject to any disclaimer, the term ofthis Patent is extended or adjusted under 35
`
`
`
`i 43583535 A 11/1982 Falkow et al. 3:13;: :31 """""""" " .... .. 435/5
`
`
`
`
`
`U-S-C- 154(1)) by 0 days-
`
`4,374,925 A
`
`2/1983 Litman etal.
`
`.... .. 435/7
`
`_
`_
`_
`_
`_
`T1115 Pawnt 1S Sublect to a lenmnal ‘115'
`Clalmen
`
`(21) APP1~ N91 08/486,070
`_
`(22) Flledi
`
`Jun- 71 1995
`
`Related U.S. AppIiCatiOII Data
`(63) Continuation of application No. 07/967,646, ?led on
`Oct. 28, 1992, now abandoned, which is a continua
`tion of application No. 07/ 607,347, ?led on Oct. 30,
`1990, now abandoned, which is a continuation of
`application NO. 07/385,986, ?led 011 Jul. 20, 1989,
`now Pat. No. 4,994,373, which is a continuation of
`application No. 06/732,374, ?led on May 9, 1985,
`now abandoned, which is a continuation-in-part of
`application No. 06/461,469, ?led on Jan. 27, 1983,
`now abandoned.
`
`(51) Int. Cl.
`(2006.01)
`C07H 21/04
`(2006.01)
`C12N 16/11
`(52) US. Cl. ................................. .. 536/243; 536/2532
`(58) Field of Classi?cation Search .................. .. 435/6,
`435/287, 810, 283.1, 285.1, 287.1, 287.2,
`435/287.7, 287.9, 288.7, 289.1, 297.1, 299.1;
`436/501; 536/221; 935/78, 77, 88; 422/50,
`422/55, 56, 57, 68.1, 69, 82.05, 82.06, 82.07,
`422/82.08
`See application ?le for complete search history.
`
`(56)
`
`References Cited
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`
`(Continued)
`
`DE
`
`FOREIGN PATENT DOCUMENTS
`P2618419
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`
`(Continued)
`
`OTHER PUBLICATIONS
`
`Manuelidis et al. (1982) Journal of Cell Biology, vol. 95, pp.
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`
`(Continued)
`Primary Examinerilohn S. Brusca
`(74) Attorney, Agent, or F irmiRonald C. Fedus
`
`(57)
`
`ABSTRACT
`
`Nucleic acids are ?xed or immobilized to non-porous solid
`supports (substrates), and include systems containing such
`supports and arrays with ?xed or immobilized nucleic acids.
`These compositions are useful for nucleic acid analyses and
`a host of applications, including, for example, detection,
`mutational analysis and quanti?cation. The non-porous solid
`supports can be transparent or translucent, and the surfaces
`can be treated with agents to ?x or immobilize the nucleic
`acids. Such agents include, for example, amine providing
`compounds, epoxy compounds and acid solutions. The ?xed
`or immobilized nucleic acids can 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
`
`Page 1 of 18
`
`HOLOGIC EXHIBIT 1001
`Hologic v. Enzo
`
`

`
`US 7,064,197 B1
`Page 2
`
`US. PATENT DOCUMENTS
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`9/1985 Dattagupta
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`5/1986 Falkow et a1.
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`4/1987 Mundy
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`8/1987 Frank
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`
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`
`5,449,767 A
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`
`FOREIGN PATENT DOCUMENTS
`
`DE
`DE
`DE
`EP
`EP
`EP
`EP
`EP
`EP
`EP
`GB
`GB
`GB
`GB
`GB
`GB
`GB
`GB
`GB
`JP
`W0
`W0
`W0
`WO
`
`12/1977
`A2724486
`10/1979
`2915082
`10/1979
`A2915082
`2/1982
`0046083
`7/1982
`0 070 687
`11/1982
`0063879 B1
`1/1983
`0070685
`1/1983
`0070687
`1/1984
`0097373 B1
`3/1984
`0103197
`7/1979
`1548741
`7/1979
`A2014727
`9/1979
`1552607
`* 10/1979
`2019408
`2019408 A 10/1979
`A2026690
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`2041922 A
`9/1980
`2045239
`10/1980
`2125946 A
`3/1984
`2825090
`11/1998
`WO 83/02276
`7/1983
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`7/1983
`W0 8302277
`7/1983
`WO8403564
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`Synthetic Organic Chemistry Chemistry), Schill, G. et al.,
`eds., Georg Thieme Verlag publishers, Stuttgart and NeW
`York, pp. 1-26, published 1980.
`ZehaVi U. et al., Journal ofOrganic Chemistry 37(14):2281
`2285 (1972).
`Seed B., Nucleic Acids Research ]O(5):1799-1810 (1982).
`
`* cited by examiner
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`

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`US 7,064,197 B1
`
`1
`SYSTEM, ARRAY AND NON-POROUS SOLID
`SUPPORT COMPRISING FIXED OR
`IMMOBILIZED NUCLEIC ACIDS
`
`CROSS-REFERENCE TO OTHER RELATED
`APPLICATIONS
`
`This is a continuation application of US. Patent Appli
`cation Ser. No. 07/967,646, ?led on Oct. 28, 1992, noW
`abandoned, Which application is a continuation application
`of US. Patent Application Ser. No. 07/607,347, ?led on Oct.
`30, 1990, also abandoned. Ser. No. 07/607,347 is a continu
`ation of US. Patent Application Ser. No. 07/385,986, ?led
`on Jul. 20, 1989, now US. Pat. No. 4,994,373 issued on Feb.
`19, 1991. Ser. No. 07/385,986 is a continuation of US.
`Patent Application Ser. No. 06/732,374, ?led on May 9,
`1985, also abandoned, Which application is a continuation
`in-part of US. Patent Application Ser. No. 06/461,469, ?led
`on Jan. 27, 1983, also abandoned.
`
`TECHNICAL FIELD OF INVENTION
`
`The present invention relates generally to the detection of
`genetic material by polynucleotide probes. More speci?
`cally, it relates to a method for quanti?ably detecting a
`targeted polynucleotide 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 quanti?able and ef?cient
`detection of genetic material is desired.
`
`BACKGROUND OF THE INVENTION
`
`In the description, the folloWing terms are employed:
`AnalyteiA substance or substances, either alone or in
`admixtures, Whose presence is to be detected and, if desired,
`quantitated. The analyte 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 analytes 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 fragments 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.
`ProbeiA 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.
`LabeliThat 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 of a signalling
`moiety, e.g., an enzyme attached directly to the sequence.
`Alternatively, the label may be a combination of a 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 quanti?able.
`Bridging MoietyiThat portion of a label Which on
`covalent attachment or non-covalent binding to a polynucle
`otide or oligonucleotide sequence acts as a link or a bridge
`betWeen that sequence and a signalling moiety.
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`Signalling MoietyiThat 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.
`SignaliThat 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 in biological 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 complementary to it. See, for example,
`T. Chard, An Introduction To Radioimmunoassay And
`Related Techniques (1978).
`Polynucleotide sequence-based detection techniques are
`characterized 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 (T), and
`guanine (G) and cytosine (C), and the detection of that
`hybridization. [M. Grunstein and D. S. Hogness, “Colony
`Hybridization: A Method For The Isolation Of Cloned
`DNAs That ContainA Speci?c Gene”, Proc. Natl. Acad. Sci.
`USA, 72, pp. 3961465 (1975)]. Such polynucleotide detec
`tion techniques can involve a ?xed analyte [see, e.g., US.
`Pat. No. 4,358,535 to FalkoW et al], or can involve detection
`of an analyte in solution [see UK. patent application 2,019,
`408 A].
`The primary recognition event of polynucleotide
`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.
`In addition to the primary recognition event, it is also
`necessary to detect When binding takes place betWeen the
`labelled polynucleotide sequence and the complementary
`sequence of the analyte. This detection is effected 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 hybridizations With suf?cient quantities of radiolabeled
`probes, the amount of radio-activity is usually directly
`proportional to the amount of analyte present. Inversely
`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.
`Ampli?cation techniques are also employed for enhanc
`ing detection Wherein the signalling event is related to the
`primary recognition event in a ratio greater than 1:1. For
`example, the signalling component of the assay may be
`
`Page 5 of 18
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`

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`US 7,064,197 B1
`
`3
`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
`non-covalently bound to the polynucleotide sequence and a
`signalling moiety that is itself detectable or that becomes
`detectable after further modi?cation.
`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 ?rst bound or attached to the sequence
`and then the signalling moiety combined With that bridging
`moiety. In addition, 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
`exempli?ed by the chemical modi?cation of the sequence
`With labels comprising radioactive moieties, ?uorescent
`moieties or other moieties that themselves provide signals to
`available detection means or the chemical modi?cation 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 of a 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 bound to an antibody, a ?uorescent moiety or
`another moiety Which is detectable by appropriate means.
`Alternatively, the bridging moiety could be a lectin, to Which
`is bound another moiety that is detectable by appropriate
`means.
`There are a Wide variety of signalling moieties and
`bridging moieties that may be employed in labels for cova
`lent attachment or non-covalent binding to polynucleotide
`sequences useful as probes in analyte detection systems.
`They include both a Wide variety of radioactive and non
`radioactive signalling moieties and a Wide variety of non
`radioactive bridging moieties. All that is required is that the
`signalling moiety provide a signal that may be detected by
`appropriate means and that the bridging moiety, if any, be
`characterized by the ability to attach covalently or to bind
`non-covalently to the sequence and also the ability to
`combine With a signalling moiety.
`Radioactive signalling moieties and combinations of vari
`ous bridging moieties and radioactive signalling moieties are
`characterized by one or more radioisotopes such as 32P, 131I,
`14C, 3 H, 60Co, 59Ni, 63Ni and the like. Preferably, the isotope
`employed emits [3 or y radiation and has a long half life.
`Detection of the radioactive signal is then, most usually,
`accomplished by means of a radioactivity detector, such as
`exposure to a ?lm.
`The disadvantages of employing a radioactive signalling
`moiety on a probe for use in the identi?cation of analytes are
`Well knoWn to those skilled in the art and include the
`precautions and hazards involved in handling radioactive
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`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 increasingly 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 non-radioactive signalling moieties also are as
`high or higher than radio-labelling techniques.
`Among the presently preferred non-radioactive signalling
`moieties or combinations of bridging/signalling moieties
`useful as non-radioactive labels are those based on the
`biotin/avidin binding system. [P. R. Langer et al., “Enzy
`matic Synthesis Of Biotin-Labeled Polynucleotides: Novel
`Nucleic Acid Af?nity Probes”, Proc. Natl. Acad. Sci. USA,
`78, pp. 6633437 (1981); J. Stavrianopoulos et al., “Glyco
`sylated DNA Probes For Hybridization/Dection Of Homolo
`gous Sequences”, presented at the Third Annual Congress
`For Recombinant DNA Research (1983); R. H. Singer and
`D. C. Ward, “Actin Gene Expression Visualized In Chicken
`Muscle Tissue Culture By Using In Situ Hybridization With
`A Biotinated Nucleotide Analog”, Proc. Natl. Acad. Sci.
`USA, 79, pp. 731435 (1982)]. For a revieW of non-radio
`active signalling and bridging/signalling systems, both
`biotin/avidin and otherWise, see D. C. Ward et al., “Modi?ed
`Nucleotides And Methods Of Preparing And Using Same”,
`European Patent application No. 63879.
`The above-referenced US. Patent Application Ser. No.
`06/255,223 Was abandoned in favor of continuation appli
`cation, US. Patent Application Ser. No. 06/496,915, ?led on
`May 23, 1983, now US. Pat. No. 4,711,955. A related
`divisional application of the aforementioned Ser. No.
`06/496,915 Was ?led (on Dec. 8, 1987) as US. Patent