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`US007064197Bl
`
`(12)
`
`United States Patent
`Rabbani et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 7,064,197 B1
`*Jun. 20, 2006
`
`4,166,103 A
`4,166,104 A
`4,228,237 A
`4,230,797 A
`4,234,563 A
`4,234,681 A
`4,251,514 A
`4,254,097 A
`4,261,893 A
`4,269,933 A
`4,271,140 A
`4,280,992 A
`4,302,204 A
`4,312,944 A
`4,318,980 A
`4,318,981 A
`4,318,982 A
`4,358,535 A
`4,374,925 A
`4,376,110 A
`4,380,580 A
`4,383,031 A
`4,391,904 A
`4,446,231 A
`4,483,920 A
`4,486,539 A
`4,516,833 A
`4,517,338 A 1
`4,537,861 A
`
`(54)
`
`(75)
`
`SYSTEM, ARRAY AND NON-POROUS SOLID
`SUPPORT COMPRISING FIXED OR
`IMMOBILIZED NUCLEIC ACIDS
`
`Inventors: Elazar Rabbani, New York, NY (US);
`Jannis G. Stavrianopoulos, Bayshore,
`NY (US); Dollie Kirtikar, Fresh
`Meadows, NY (US); Kenneth H.
`Johnston, New Orleans, LA (US);
`Barbara E. Thalenfeld, New York, NY
`(US)
`
`(73)
`
`Assignee: Enzo Life Sciences, Inc. c/0 Enzo
`Biochem, Inc., Farmingdale, NY (US)
`
`(*)
`
`Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35 V
`U.S.C, 154(b) by 0 days.
`
`This patent is subject to a terminal dis-
`claimer.
`‘
`
`(21)
`
`Appl. No.: 08/486,070
`
`(22)
`
`Filed:
`
`Jun. 7, 1995
`
`(63)
`
`(51)
`
`(52)
`(58)
`
`(56)
`
`Related U.S. Application Data
`
`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.
`
`Int. Cl.
`(2006.01)
`C07H 21/04
`(2006.01)
`C12N 16/11
`U.S. C1.
`................................. .. 536/24.3; 536/25.32
`Field of Classification 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 file for complete search history.
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`....................... .. 195/63
`6/1972 Miller
`....................... .. 195/63
`2/1973 Miller
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`4/1976 Bomstein et a1. ............ .. 424/1
`1/1977 Barrett
`........ ..
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`
`
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`
`(Continued)
`FOREIGN PATENT DOCUMENTS
`P2618419
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`
`DE
`
`(Continued)
`OTHER PUBLICATIONS
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`Manuelidis et al. (1982) Journal of Cell Biology, vol. 95, pp.
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`
`(Continued)
`
`Primary Examiner-—.lohn S. Brusca
`(74) Attorney, Agent, or Firm—Ronald C. Fedus
`
`(57)
`
`ABSTRACT
`
`Nucleic acids are fixed or immobilized to non-porous solid
`supports (substrates), and include systems containing such
`supports and arrays with fixed or immobilized nucleic acids.
`These compositions are useful for nucleic acid analyses and
`a host of applications, including, for example, detection,
`mutational analysis and quantification. The non-porous solid
`supports can be transparent or translucent, and the surfaces
`can be treated with agents to fix or immobilize the nucleic
`acids. Such agents include, for example, amine providing
`compounds, epoxy compounds and acid solutions. The fixed
`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—strandcd.
`
`238 Claims, No Drawings
`
`BD Exhibit 1001
`
`8/1979 Wagner et al. ............... .. 424/l
`8/1979 Wagner et al.
`..
`
`10/1980 Hevey et a1.
`10/1980 Bogislaskietal. .......... .. 435/7
`11/1980 Rippe
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`2/1981 Rippe
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`4/1981 Boguslaski et al.
`5/1981 Pazos
`.... .. 424/1
`6/1981 Bunting ................
`424/1
`7/1981 Sugiura et al.
`.
`23/230.3
`11/1981 Wahl et al.
`...... ..
`435/7
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`435/7
`3/1982 Boguslaskiet al.
`435/7
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`...... ..
`435/7
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`..
`...... .. 435/5
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`..
`435/7
`2/1983 Litman et al.
`..
`.. 436/513
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`. . . .. 435/7
`4/1983 Boguslaski et al.
`. . . .. 435/7
`5/1983 Boguslaskietal.
`. . . .. 435/7
`7/1983 Litman et al.
`. . . . .
`435/7
`5/1984 Self ............ ..
`‘
`435/6
`11/1984 Gillespie et a1.
`12/1984 Ranki et a1.
`.............. .. 436/504
`5/1985 Fusek
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`8/1985 Elings
`
`
`
`. . . .
`. . . .
`
`....... .. 260/326
`
`BD Exhibit 1001
`
`

`
`US 7,064,197 B1
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`........... .. 435/6
`
`................. .. 536/29
`
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`
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`5,260,433 A
`
`...... ..
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`‘
`,
`Amit B. et'al., Journal ofOganic Chemistry 39(2): 192-196
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`(1974).
`Barltrop J .A. et al., Chemical Communications 22:822-823
`(1966).
`.
`'
`Flanders D.C. and Smith, H.l., Applied Physics Letters
`3I(7):426-428 (1977).
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`Ohtsuka E. et al., Nucleic Acids Research I(l0):1351-1357
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`Patchornik A. and Amit B., Journal ofthe American Chemi-
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`_
`Pillai V.N.R., Synthesis (International Journal of Methods in
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`Seed B., NucleicAcids Research ]0(5):1799-1810 (1982).
`
`* cited by examiner
`
`

`
`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 U.S. Patent Appli-
`cation Ser. No. 07/967,646, filed on Oct. 28, 1992, now
`abandoned, which application is a continuation application
`ofU.S. Patent Application Ser. No. 07/607,347, filed on Oct.
`30, 1990, also abandoned. Ser. No. 07/607,347 is a continu-
`ation of U.S. 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 US.
`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.
`
`TECHNICAL FIELD OF INVENTION
`
`5
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`10
`
`15
`
`20
`
`The present invention relates generally to the detection of
`genetic material by polynucleotide probes. More specifi-
`cally, it relates to a method for quantifiably detecting a 25
`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 30
`medical applications wherein quantifiable and efficient
`detection of genetic material is desired.
`
`BACKGROUND OF THE INVENTION
`
`35
`
`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 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 techniquesare 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 Specific Gene”, Proc. Natl. Acad. Sci.
`USA, 72, pp. 3961-65 (l975)]. Such polynucleotide detec-
`tion techniques can involve a fixed analyte [see, e.g., U.S.
`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 aprecise 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 elfected 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 suflicient 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.
`Amplification 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
`
`In the description, the following terms are employed:
`Analyte--A 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 40
`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.
`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 of a signalling
`moiety, eg., 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 quantifiable.
`Bridging Moiety—That 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.
`
`45
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`50
`
`55
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`60
`
`65
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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 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. 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
`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 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 ofthe
`polynucleotide sequence may be a bridging moiety non-
`covalently.bound to an antibody,
`ab fluorescent 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, 13 11,
`“C, 3H, 5°Co, 59Ni, “Ni and the like. Preferably, the isotope
`employed emits B or 7 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 film.
`The disadvantages of employing a radioactive signalling
`moiety on a probe for use in the identification of analytes are
`Well known to those skilled in the art and include the
`precautions and hazards involved in handling radioactive
`
`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 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 Affinity Probes”, Proc. Natl. Acad. Sci. USA,
`78, pp. 6633—37 (1981); J. Stavrianopoulos et al., “Glyco-
`sylated DNA Probes For Hybridization/Dection Of Homolo-
`gous Sequences”, presented at the Third Armual 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. 733l~35 (l982)]. For a review of non-radio-
`active signalling and bridging/signalling systems, both
`biotin/avidin and otherwise, see D. C. Ward et al., “Modified
`Nucleotides And Methods Of Preparing And Using Same”,
`European 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 U.S. Pat. No; 4,711,955. A related
`divisional application of the aforementioned Ser. No.
`06/496,915 was filed (on Dec. 8, 1987) as U.S. 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 of U.S. Patent Application
`Ser. No. 07/140,980, filed on Jan. 5, 1988, the latter now
`abandoned. Two divisional applications of the afor