United States Patent
`
`[19]
`
`6,143,496
`Nov. 7, 2000
`Brown et al.
`[45] Date of Patent:
`
`[11] Patent Number:
`
`US006143496A
`
`[54] METHOD OF SAMPLING, AMPLIFYING
`AND QUANTIFYING SEGMENT OF
`NUCLEIC ACID, POLYMERASE CHAIN
`REACTION ASSEMBLY HAVING
`NANOLITER-SIZED SAMPLE CHAMBERS,
`AND METHOD OF FILLING ASSEMBLY
`
`[75]
`
`Inventors: James F. Brown, Clifton, Va.;
`Jonathan E. Silver, Bethesda, Md.;
`Olga V. Kalinina, Toronto, Canada
`
`[73] Assignees: Cytonix Corporation, Beltsville, Md.;
`The United States of America as
`
`represented by the Department of
`Health and Human Services,
`Washington, DC.
`
`[21] Appl. No.2 08/838,262
`
`[22]
`
`Filed:
`
`Apr.17, 1997
`
`Int. C1.7 ......................... G01N 33/543, G01N 33/68
`[51]
`[52] U.S.Cl. ........................ 435/6; 435/287.1; 435/2872;
`435/2883; 435/288.7; 436/164; 436/172;
`436/518; 436/524; 436/527; 436/531; 436/805;
`436/809; 422/58
`[58] Field of Search ......................... 435/6, 287.1, 287.2,
`435/2883, 288.7; 436/164, 172, 518, 524,
`527, 531, 805, 809; 422/58
`
`[56]
`
`References Cited
`
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`
`5/1986 Britten et a1.
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`.
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`9/1994 Stapleton et a1.
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`1/1995 Sutton et a1.
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`3/1996 Wilding et al.
`.
`5,498,392
`4/1996 Brown .
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`6/1996 Drmanac et a1.
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`5,585,069 12/1996 Zanzucchi et a1.
`
`.
`
`.
`
`436/94
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`OTHER PUBLICATIONS
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`Wittwer et al., “The LightcyclerTM: A Microvolume Multi-
`sample Flourimeter With Rapid Temperature Control”; Bio
`Techniques, vol. 22, No. 1, pp. 176—181 (Jan. 1997).
`Woolley et al., “Ultra—High—Speed DNA Fragment Separa-
`tions Using Microfabricated Capillary Electrophoresis
`Chips”, Proc. Natl. Acad. Sci. USA, vol.
`91, pp.
`11348—11352, Biophysics, (1994).
`Wilding et al., PCR in a Silicone Mircrostructure; Clinical
`Chemistry, vol. 40, No. 9, pp. 1815—1818 (1994).
`Good et al., Generalization of Theory for Estimation of
`Interfacial Energies; Chemistry and Physics of Interfaces,
`ACS, pp. 91—96 (1971).
`Burns et al., Microfabricated Structures for Integrated DNA
`Analysis; Proc. Natl. Acad Sci. USA, vol. 93, pp.
`5556—5561, Genetics (1996).
`Rigler, “Fluorescence Correlations, Single Molecule Detec-
`tion and Large Number Screening Applications in Biotech-
`nology”; Journal of Biotechnology, vol. 41, pp. 177—186
`(1995).
`
`(List continued on next page.)
`
`Primary Examiner—Christopher L. Chin
`Attorney, Agent, or Firm—Kilyk & Bowersox, P.L.L.C.
`
`[57]
`
`ABSTRACT
`
`Methods of detecting and quantifying genomic nucleic acid
`molecule sequences are provided using the simultaneous
`amplification of a plurality of discrete nanoliter-sized
`samples. A miniaturized closed assembly is also provided
`for carrying out amplification of a nucleic acid molecule by
`polymerase chain reaction in multiple nanoliter-sized
`samples. Methods of filling miniaturized sample chambers
`are also provided as are methods for determining the number
`of template molecules in a sample by conducting replicate
`nucleic acid sequence amplification reactions on a set of
`terminally diluted samples and counting the number of
`positive amplification reactions. The methods can be used to
`detect a single starting nucleic acid target molecule.
`
`17 Claims, 7 Drawing Sheets
`
`
`
`Ambry Exhibit 1010
`
`Ambry Exhibit 1010
`
`

`

`6,143,496
`Page 2
`
`OTHER PUBLICATIONS
`
`Cheng et al.; “Analysis of Ligase Chain Reaction Products
`Amplified in a Silicon—Glass Chip Using Capillary Electro-
`phoresis”, Journal of Chromatography, vol. 732, pp.
`151—158 (1996).
`Kricka et al.; “Imaging of Chemiluminescent Reactions in
`Mesocale Silicon—Glass Microstructures”; J. Biolumin
`Chemilumin, vol. 9, pp. 135—138 (1994).
`Woolley et al., “Ultra—High—Speed DNA Sequencing Using
`Capillary Electrophoresis Chips”, Anal—Chem, vol. 67, No.
`20, pp. 3676—3680 (1995).
`Hawkins et al.; “Incorporation of a Fluorescent Guanosine
`Analog into Oligonucleotides and its Application to a Real
`Time Assay for the HIV—1 Integrase 3‘—Processing Reac-
`tion”; Nucleic Acids Research, vol. 23, No. 15, pp.
`2872—2880 (1995).
`Tyagi et al.; “Molecular Beacons: Probes That Fluoresce
`Upon Hybridization” Nature Biotechnology, vol. 14, pp.
`303—308 (1996).
`Holland et al.; Detection of Specific Polymerase Chain
`Reaction Product by Utilizing the 5‘93‘Exonuclease Activ-
`ity of Thermus Aquaticus DNA Polymerase; Proc. Natl.
`Acad. Sci., vol. 88, Biochemistry, pp. 7276—7280 (1991).
`Livak et al.; “Oligonucleotides with Fluorescent Dyes at
`Opposite Ends Provide a Quenched Probe System Useful for
`Detecting PCR Product and Nucleic Acid Hybridization”;
`PCR Method and Applications, pp. 357—362 (1995).
`Sninsky et al.; “The Application of Quantitative Polymerase
`Chain Reaction to Therapeutic Monitoring”; AIDS, vol. 7
`(Supp 2), pp. S29—S34 (1993).
`Becker—Andre et al., Absolute mRNA Quantification Using
`the Polymerase Chain Reaction (PCR); Nucleic Acids
`Research, vol. 17, No. 22, pp. 9437—9446 (1989).
`Gilliland et al.; “Analysis of Cytokine mRNA and DNA:
`Detection and Quantitation by Competitive Polymerase
`Chain Reaction”, Proc. Natl. Acad. Sci. USA, vol. 87,
`Genetics, pp. 2725—2729 (1990).
`Higuchi et al.; “Simultaneous Amplification and Detection
`of Specific DNA Sequences”; Biotechnology, vol. 10, pp.
`413—417 (1992).
`
`Heid et al.; “Real Time Quantitative PCR”, Genome
`Research, No. 6, pp. 986—994 (1996).
`Gibson et al.; “A Novel Method for Real Time Quantitative
`RT—PCR”; Genome Research, No. 6, pp. 995—1001 (1996).
`Gerard et al.; “A Rapid and Quantitative Assay to Estimate
`Gene Transfer into Retrovirally Transduced Hematopoietic
`Stem/Progenitor Cells Using a 96—Well Format PCR and
`Fluorescent Detection System Universal for MMLV—Based
`Proviruses”; Human Gene Therapy, No. 7, pp. 343—354
`(1996).
`Wittwer et al., “Rapid Cycle DNAAmplification”, Biotech-
`niques, vol. 10, No. 1, pp. 76—83 (1991).
`Chang, Physical Chemistry With Applications to Biological
`Systems, 2”“ Edition, Sec. 5.4, p. 87.
`Berg, Random Walks in Biology, “Diffusion: Microscopic
`Theory”, pp. 10, 49 (1983).
`Burns et al.; Microfabricated Structures for Integrated DNA
`Analysis, Proc. Natl. Acad Sci., vol. 93, Genetics, pp.
`5556—5561 (1996).
`Cheng et al.; Chip PCR.II. Investigation of Different PCR
`Amplification Systems in Microfabricated Silicon—Glass
`Chips; NucleicAcids Research, vol. 24, No. 2, pp. 380—385
`(1996).
`Woolley et al.; “Functional Integration of PCR Amplifica-
`tion and Capillary Electrophoresis in a Microfabricated
`DNAAnalysis Device”;Anal. Chem. No. 68, pp. 4081—4086
`(1996).
`Wittwer et al.; Continuous Fluorescence Monitoring of
`Rapid Cycle DNA Amplification; Biotechniques, 22, pp.
`130—138 (Jan. 1997).
`Hawkins et al.; Fluorescence Properties of Pteridine Nucleo-
`side Analogs as Monomers and Incorporated into Oligo-
`nucleotides, Analytical Biochemistry, 244, pp.
`86—95
`(1997).
`Xu et al., “Direct Measurement of Single—Molecule Diffu-
`sion and Photodecomposition in Free Solution”, Science,
`vol. 275, pp. 1106—1109 (Feb. 1997).
`CRC Handbook of Chemistry and Physics, 74th Edition,
`Lide (Editor—In—Chief), p. 6—10 (1993—1994).
`
`Ambry Exhibit 1010
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`Ambry Exhibit 1010
`
`

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`US. Patent
`
`Nov. 7, 2000
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`Sheet 1 0f 7
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`6,143,496
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`Ambry Exhibit 1010
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`Ambry Exhibit 1010
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`

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`US. Patent
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`Nov. 7, 2000
`
`Sheet 2 0f 7
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`6,143,496
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`Ambry Exhibit 1010
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`Ambry Exhibit 1010
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`US. Patent
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`Nov. 7, 2000
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`Sheet 4 0f 7
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`6,143,496
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`US. Patent
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`Nov. 7, 2000
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`Sheet 5 0f 7
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`6,143,496
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`US. Patent
`
`Nov. 7, 2000
`
`Sheet 7 0f 7
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`6,143,496
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`Ambry Exhibit 1010
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`Ambry Exhibit 1010
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`

`

`6,143,496
`
`1
`
`METHOD OF SAMPLING, AMPLIFYING
`AND QUANTIFYING SEGMENT OF
`NUCLEIC ACID, POLYMERASE CHAIN
`REACTION ASSEMBLY HAVING
`NANOLITER-SIZED SAMPLE CHAMBERS,
`AND METHOD OF FILLING ASSEMBLY
`
`GOVERNMENT RIGHTS
`
`Part of the work leading to this invention was carried out
`with the United States Government support provided under
`the National Institutes of Health CRADA contract No.
`A1000079. Therefore, the United States Government has
`certain rights in and to the present invention.
`
`FIELD OF THE INVENTION
`
`The present invention relates to the in vitro amplification
`of a segment of nucleic acid, methods to analyze concen-
`trations of specific nucleic acids in sample fluids, and
`methods for detecting amplification of a target nucleic acid
`sequence. The present invention also relates to miniaturized
`analytical assemblies and methods of filling miniaturized
`analytical assemblies.
`
`BACKGROUND OF THE INVENTION
`
`Nucleic acid amplification techniques such as polymerase
`chain reaction (PCR), ligase chain reaction (LCR), strand
`displacement amplification (SDA), and self-sustained
`sequence replication (3SR) have had a major impact on
`molecular biology research. In particular, PCR, although a
`relatively new technology, has found extensive application
`in various fields including the diagnosis of genetic disorders,
`the detection of nucleic acid sequences of pathogenic organ-
`isms in clinical samples, the genetic identification of foren-
`sic samples, and the analysis of mutations in activated
`oncogenes. In addition, PCR amplification is being used to
`carry out a variety of tasks in molecular cloning and analysis
`of DNA. These tasks include the generation of specific
`sequences of DNA for cloning or use as probes, the detection
`of segments of DNA for genetic mapping, the detection and
`analysis of expressed sequences by amplification of particu-
`lar segments of cDNA, the generation of libraries of cDNA
`from small amounts of mRNA,
`the generation of large
`amounts of DNA for sequencing, the analysis of mutations,
`and for chromosome crawling. During the next few years,
`PCR, other amplification methods, and related technologies
`are likely to find increasing application in many other
`aspects of molecular biology.
`Unfortunately, problems exist in the application of PCR to
`clinical diagnostics. Development has been slow due in part
`to:
`labor intensive methods for detecting PCR product;
`susceptibility of PCR to carryover contamination—false
`positives due to contamination of a sample with molecules
`amplified in a previous PCR; and difficulty using PCR to
`quantitate the number of target nucleic acid molecules in a
`sample. A need exists for a simple method of quantitative
`analysis of target nucleic acid molecules in a sample.
`Recently, significant progress has been made in overcom-
`ing some of the problems of clinical diagnostic nucleic acid
`amplification through the development of automatable
`assays for amplified product that do not require that the
`reaction vessel be opened, thereby minimizing the risk of
`carryover contamination. Most of these assays rely on
`changes in fluorescent light emission consequent to hybrid-
`ization of a fluorescent probe or probes to amplified nucleic
`acid. One such assay involves the hybridization of two
`
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`probes to adjacent positions on the target nucleic acid. The
`probes are labeled with different fluors with the property that
`energy transfer from one fluor stimulates emissions from the
`other when they are brought together by hybridization to
`adjacent sites on the target molecule.
`Another assay, which is commercially available, is the
`“TaqMan” fluorescence energy transfer assay and kit, avail-
`able from Perkin Elmer, Applied Biosystems Division, Fos-
`ter City, Calif. This type of assay is disclosed in the
`publication of Holland et al., Detection of specific Poly-
`merase chain reaction product by utilizing the 5'93’ exo-
`nuclease activity of Thermus aquaticus DNA polymerase,
`Proc. Natl. Acad. Sci. USA, Vol. 88, pp. 7276—7280, August
`1991, and in the publication of Livak et al., Oligonucleotides
`with Fluorescent Dyes at Opposite Ends Provide a
`Quenched Probe System Useful for Detecting PCR Product
`and Nucleic Acid Hybridization, PCR Methods and Applic.,
`4, pp. 357—362 (1995). The “TaqMan” or 5' exonuclease
`assay uses a single nucleic acid probe complementary to the
`amplified DNA and labeled with two fluors, one of which
`quenches the (other. If PCR product is made,
`the probe
`becomes susceptible to degradation via an exonuclease
`activity of Taq polymerase that is specific for DNA hybrid-
`ized to template (“TaqMan” activity). Nucleolytic degrada-
`tion allows the two fluors to separate in solution which
`reduces quenching and increases the intensity of emitted
`light of a certain wavelength. Because these assays involve
`fluorescence measurements that can be performed without
`opening the amplification vessel, the risk of carryover con-
`tamination is greatly reduced. Furthermore, the assays are
`not labor intensive and are easily automated.
`The TaqMan and related assays have provided new ways
`of quantitating target nucleic acids. Early methods for quan-
`titation relied on setting up amplification reactions with
`known numbers of target nucleic acid molecules and com-
`paring the amount of product generated from these control
`reactions to that generated from an unknown sample, as
`reviewed in the publication by Sninsky et al. The application
`of quantitative polymerase chain reaction to therapeutic
`monitoring, AIDS 7 (SUPPL. 2), PP. S29—S33 (1993). Later
`versions of this method used an “internal control”, i.e., a
`target nucleic acid added to the amplification reaction that
`should amplify at the same rate as the unknown but which
`could be distinguished from it by Virtue of a small sequence
`difference, for example, a small insertion or deletion or a
`change that led to the gain or loss of a restriction site or
`reactivity with a special hybridization probe, as disclosed in
`the publication by Becker-Andre, et al., Absolute mRNA
`quantification using the polymerase chain reaction (PCR). A
`novel approach by a PCR aided transcript titration assay
`(PATTY), Nucleic Acids Res., Vol. 17, No. 22, pp.
`9437—9446 (1989), and in the publication of Gilliland et al.,
`Analysis of cytokine mRNA and DNA: Detection and quan-
`titation by competitive polymerase chain reaction, Proc.
`Natl. Acad. Sci. USA, Vol. 87, pp. 2725—2729 (1990). These
`methods have the disadvantage that slight differences in
`amplification efficiency between the control and experimen-
`tal nucleic acids can lead to large differences in the amounts
`of their products after the million-fold amplification char-
`acteristic of PCR and related technologies, and it is difficult
`to determine relative amplification rates accurately.
`Newer quantitative PCR methods use the number of
`cycles needed to reach a threshold amount of PCR product
`as a measure of the initial concentration of target nucleic
`acid, with DNA dyes such as ethidium bromide or SYBRTM
`Green I, or “TaqMan” or related fluorescence assays used to
`follow the amount of PCR product accumulated in real time.
`
`Ambry Exhibit 1010
`
`Ambry Exhibit 1010
`
`

`

`6,143,496
`
`3
`Measurements using ethidium bromide are disclosed in the
`publication of Higuchi et al., Simultaneous Amplification
`and Detection of Specific DNA Sequences, BIO/
`TECHNOLOGY, Vol. 10, pp. 413—417 (1992). “TaqMan”
`assays used to follow the amount of PCR product accumu-
`lated in real time are disclosed in the publication of Heid et
`al., Real Time Quantitative PCR, Genome Research, Vol. 6,
`pp. 986—994 (1996), and in the publication of Gibson et al.,
`A Novel Method for Real Time Quantitative RT-PCR,
`Genome Research, Vol. 6, pp. 995—1001 (1996). However,
`these assays also require assumptions about relative ampli-
`fication efficiency in different samples during the exponen-
`tial phase of PCR.
`An alternative method of quantitation is to determine the
`smallest amount of sample that yields PCR product, relying
`on the fact that PCR can detect a single template molecule.
`Knowing the average volume of sample or sample dilution
`that contains a single target molecule, one can calculate the
`concentration of such molecules in the starting sample.
`However, to accumulate detectable amounts of product from
`a single starting template molecule usually requires that two
`or more sequential PCRs have to be performed, often using
`nested sets of primers, and this accentuates problems with
`carryover contamination.
`Careful consideration of the factors affecting sensitivity to
`detect single starting molecules suggests that decreasing the
`volume of the amplification reaction might improve sensi-
`tivity. For example,
`the “TaqMan” assay requires near
`saturating amounts of PCR product
`to detect enhanced
`fluorescence. PCRs normally saturate at about 1011 product
`molecules/microliter (molecules/n1) due in part
`to rapid
`reannealing of product strands. To reach this concentration
`of product after 30 cycles in a 10 a1 PCR requires at least 103
`starting template molecules (103x230/10 #131011/yl). Some-
`what less than this number of starting molecules can be
`detected by increasing the number of cycles, and in special
`circumstances even single starting molecules may be detect-
`able as described in the publication of Gerard et al., A Rapid
`and Quantitative Assay to Estimate Gene Transfer into
`Retrovirally Transa'ucea' Hematopoietic Stem/Progenitor
`Cells Using a 96-Well Format PCR and Fluorescent Detec-
`tion System Universal for MMLV—Basea' Proviruses, Human
`Gene Therapy, Vol. 7, pp. 343—354 (1996). However, this
`strategy usually fails before getting to the limit of detecting
`single starting molecules due to the appearance of artifactual
`amplicons derived from the primers (so called “primer-
`dimers”) which interfere with amplification of the desired
`product.
`If the volume of the PCR were reduced 1000-fold to ~10
`
`nanoliters (n1), then a single round of 30 cycles of PCR
`might suffice to generate the saturating concentration of
`product needed for detection by the TaqMan assay, i.e. 1><23O
`per 10 nanoliters lell per microliter. Attempts have been
`made to miniaturize PCR assemblies but no one has devel-
`
`oped a cost-effective PCR assembly which can carry out
`PCR in a nanoliter-sized sample. Part of the problem with
`miniaturization is that evaporation occurs very rapidly with
`small sample volumes, and this problem is made worse by
`the need to heat samples to ~90° C. during thermocycling.
`In addition to potential advantages stemming from ability
`to detect single target nucleic acid molecules, miniaturiza-
`tion might also facilitate the performance of multiple dif-
`ferent amplification reactions on the same sample. In many
`situations it would be desirable to test for the presence of
`multiple target nucleic acid sequences in a starting sample.
`For example, it may be desirable to test for the presence of
`multiple different viruses such as HIV-1, HIV-2, HTLV—1,
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`HBV and HCV in a clinical specimen; or it may be desirable
`to screen for the presence of any of several different
`sequence variants in microbial nucleic acid associated with
`resistance to various therapeutic drugs; or it may be desir-
`able to screen DNA or RNA from a single individual for
`sequence variants associated with different mutations in the
`same or different genes, or for sequence variants that serve
`as “markers” for the inheritance of different chromosomal
`segments from a parent. Amplification of different nucleic
`acid sequences and/or detection of different sequence vari-
`ants usually requires separate amplification reactions with
`different sets of primers and/or probes. If different primer/
`probe sets were positioned in an array format so that each
`small region of a reaction substrate performed a different
`amplification/detection reaction, it is possible that multiple
`reactions could be carried out in parallel, economizing on
`time, reagents, and volume of clinical specimen.
`A need therefore exists for a device that can form and
`
`retain a sample volume of about 10 nanoliters or less and
`enable amplification to be performed without significant
`evaporation. A need also exists for a reliable means of
`detecting a single starting target nucleic acid molecule to
`facilitate quantification of target nucleic acid molecules. A
`need also exists for performing multiple different amplifi-
`cation and detection reactions in parallel on a single speci-
`men and for economizing usage of reagents in the process.
`
`SUMMARY OF THE INVENTION
`
`According to the present invention, methods and appara-
`tus for performing nucleic acid amplification on a miniatur-
`ized scale are provided that have the sensitivity to determine
`the existence of a single target nucleic acid molecule. The
`invention also provides analytical assemblies having sample
`retaining means which form, isolate and retain fluid samples
`having volumes of from about one microliter to about one
`picoliter or less. The invention also provides a method of
`forming fluid samples having sample volumes of from about
`one microliter to about ones picoliter or less, and retaining
`the samples under conditions for thermocycling. The inven-
`tion also provides an analytical assembly having means to
`determine simultaneously the presence in a sample of mul-
`tiple different nucleic acid target molecules.
`According to embodiments of the invention, PCR condi-
`tions are provided wherein a single target nucleic acid
`molecule is confined and amplified in a volume small
`enough to produce a detectable product through fluorescence
`microscopy. According to embodiments of the invention,
`samples of a few nanoliters or less can be isolated, enclosed
`and retained under thermocycling conditions, and a plurality
`of such samples can be collectively analyzed to determine
`the existence and initial concentration of target nucleic acid
`molecules and/or sequences. According to some embodi-
`ments of the invention, sample retaining chambers having
`volumes of about 10 picoliters or less can be achieved.
`According to embodiments of the invention, methods of
`forming small fluid samples, isolating them and protecting
`them from evaporation are provided wherein different affini-
`ties of a sample retaining means and a communicating
`channel are used to retain sample in the means while a
`second fluid displaces sample from the channel. According
`to some embodiments of the invention, the resultant isolated
`samples are then subject to PCR thermal cycling.
`According to embodiments of the invention, methods are
`provided for determining the existence and/or initial con-
`centration of a target nucleic acid molecule in samples of
`about 1 microliter or less. According to some embodiments
`
`Ambry Exhibit 1010
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`Ambry Exhibit 1010
`
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`

`6,143,496
`
`5
`of the invention, methods are provided for a clinical diag-
`nosis PCR analysis which can quickly and inexpensively
`detect a single target nucleic acid molecule.
`According to some embodiments of the invention, sample
`chambers of about 1 microliter or less are provided that have
`a greater affinity for a sample to be retained than for a
`displacing fluid. The displacing fluid displaces sample from
`around the chambers and isolates the sample portion
`retained in the chambers.
`
`According to embodiments of the invention, nucleic acid
`samples are isolated, retained and amplified in microcapil-
`lary devices having volumes of about 100 nanoliters or less,
`including microcapillary tubes, planar microcapillaries and
`linear microcapillaries. The devices may be provided with
`absolute, selective or partial barrier means.
`According to embodiments of the invention, a porous or
`microporous material retains samples of about 100 nanoli-
`ters or less, and an assembly is provided which includes a
`cover for sealing sample within the porous or microporous
`material.
`
`According to embodiments of the present invention, PCR
`methods and apparatus are provided wherein the sensitivity
`of a “TaqMan” fluorescence assay can be used to enable
`detection of single starting nucleic acid molecule in reaction
`volumes of about 100 nl or less. According to the present
`invention, assemblies for retaining PCR reaction volumes of
`about 10 n1 or less are provided, wherein a single target
`molecule is sufficient to generate a fluorescence-detectable
`concentration of PCR product.
`According to the invention, methods are provided for
`carrying out PCR in minute volumes, for example, 10 n1 or
`less, which allows detection of PCR products generated
`from a single target molecule using the “TaqMan” or other
`fluorescence energy transfer systems.
`invention,
`According to embodiments of the present
`methods of detecting and quantifying DNA segments by
`carrying out polymerase chain reaction in a plurality of
`discrete nanoliter-sized samples are provided. The present
`invention also provides methods for determining the number
`of template molecules in a sample by conducting replicate
`polymerase chain reactions on a set of terminally diluted or
`serially smaller samples and counting the number of positive
`polymerase chain reactions yielding specific product. The
`present invention is useful in detecting single starting mol-
`ecules and for quantifying the concentration of a nucleic
`acid molecule in a sample through PCR. The present inven-
`tion also provides methods of detecting and quantifying a
`plurality of target DNA sequences.
`The present invention also provides methods and assem-
`blies for separating and/or analyzing multiple minute por-
`tions of a sample of fluid medium that could be useful for
`other applications. Applications of the apparatus of the
`invention include the separation of biological samples into
`multiple minute portions for the individual analysis of each
`portion, and can be used in the fields of fertility,
`immunology, cytology, gas analysis, and pharmaceutical
`screening.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`The invention will be described in connection with vari-
`
`ous embodiments exemplified in the accompanying
`drawings, wherein:
`FIG. 1 is an exploded view of an analytical assembly
`according to an embodiment of the present invention, shown
`in partial cutaway;
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`4s
`
`50
`
`55
`
`60
`
`65
`
`6
`FIG. 2 is an exploded view of an analytical assembly
`according to an embodiment of the present invention, shown
`in partial cutaway, comprising sample chambers in the form
`of wells formed into patterned layers on the inner surfaces
`of both a top plate and a bottom plate;
`FIG. 3 is a cross-sectional view through a longitudinal
`central portion of an analytical assembly according an
`embodiment of the present invention;
`FIG. 4 is a perspective view of a bottom portion of an
`analytical assembly according to an embodiment of the
`present invention,
`the bottom portion comprising sample
`retaining means in the form of patches of fluid retaining
`material formed on a patterned layer coated on the inner
`surface of a bottom plate;
`FIG. 5 is a cross-sectional view through a longitudinal
`central portion of an analytical assembly according an
`embodiment of the present invention;
`FIG. 6A is a perspective View of a bottom portion of an
`analytical assembly according to another embodiment of the
`present invention;
`FIG. 6B is an enlarged view of portion VIA shown in FIG.
`6A;
`FIG. 7 is a top plan view of a microcapillary analytical
`assembly according to an embodiment of the present inven-
`tion;
`FIG. 8 is a histogram showing the maximum values of the
`fluoresceinzrhodamine intensity ratio in over 100 capillary
`reactions of terminally diluted genomic DNA carried out in
`an assembly according to the present invention and accord-
`ing to a method according to the invention; and
`FIGS. 9—14 are plots of the fluoresceinzrhodamine ratio
`along a few representative microcapillaries containing
`samples subject
`to PCR in accordance with the present
`invention.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`invention,
`According to embodiments of the present
`methods of manipulating a sample of fluid medium are
`provided. The methods comprise loading a sample of fluid
`medium into sample retaining means of an analytical assem-
`bly and displacing excess sample from areas adjacent to the
`portion retained by the sample retaining means. According
`to embodiments of the invention, sample fluid is displaced
`from regions adjacent to the retained sample, without dis-
`placing the retained sample. In some embodiments, a dis-
`placing fluid is used to isolate a retained sample, and the
`displacing fluid may be curable to form a retaining chamber
`entrapping the fluid sample retained by the sample retaining
`means.
`
`The assemblies of the present invention provide samples
`or sample portions enclosed in a protective environment
`which protects the sample or portion from evaporation and
`contamination. Preferably, sample is protected from evapo-
`ration at temperatures of about 95° C. or more, for example,
`at temperatures achieved during thermal cycling under con-
`ditions for PCR. The isolated, entrapped or enclosed sample
`or portion is preferably protected from contamination and
`evaporation throughout an amplification protocol,
`for
`example, a PCR thermal cycling protocol.
`According to some embodiments of the invention, an
`analytical assembly is provided and comprises a plurality of
`sample chambers each confined in at least one dimension by
`opposing barriers separated by a first dimension of about
`500 microns or less, preferably by 100 microns or less, and
`
`Ambry Exhibit 1010
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`

`6,143,496
`
`7
`in some embodiments by about 20 microns. Means are
`provided for sealing the plurality of sample chambers to
`prevent evaporation and contamination of fluid sample con-
`fined within the plurality of sample chambers. Means are
`provided for restraining reaction product formed from reac-
`tions of a chemical substance restrained within the plurality
`of sample chambers. According to some embodiments,
`means may be provided for minimizing diffusion and sub-
`stantially preventing convection of amplification reaction
`product formed from reactions of the fluid sample restrained
`within the plurality of sample chambers. If provided, at least
`one of the means for restraining and the means for mini-
`mizing diffusion and substantially preventing convection
`may preferably comprise a patterned layer which at least
`partially defines the plurality of sample chambers.
`Preferably,
`the fluid sample contains at
`least one target
`nucleic acid molecule to be amplified and constituents for
`enabling amplification of the target nucleic acid molecule.
`The fluid sample is divined into a plurality of sample
`portions and the plurality of sample chambers are loaded
`which respective portions of the fluid sample. According to
`some embodiments of the invention, the sample portions are
`in fluid communication with each other, rather than being
`completely isolated from each other, and separated by
`barrier means which may be in the shape of crosses, lines,
`semicircles, circles having at least one opening along the arc
`thereof, or other geometric shapes. According to embodi-
`ments of the invention, the barrier means may define sample
`retaining portions or chambers of the assembly. According to
`some embodiments, means for minimizing diffusion and
`substantially preventing convection are provided, and may
`comprise the herein described barrier means. According to
`some embodiments,
`the barrier means includes physical
`structures which may extend between the aforementioned
`opposing barriers which are separated by 500 microns. The
`barrier means may form a wall or walls between the oppos-
`ing barriers. The barrier means may comprise flow restric-
`tion means. Flow restriction means may be, for example,
`semi-circular walls extending from one of the opposing
`barriers toward the other, and having the concave side of the
`semi-circle facing the direction of fluid flow during loading,
`and the semicircular arc may have at least hole or interrup-
`tion therein through which air may esca