United States Patent [19J
`Brown et al.
`
`[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, D.C.
`
`[21] Appl. No.: 08/838,262
`
`[22]
`
`Filed:
`
`Apr. 17, 1997
`
`[51]
`[52]
`
`[58]
`
`[56]
`
`Int. Cl.7 ......................... GOlN 33/543; GOlN 33/68
`U.S. Cl. ........................ 435/6; 435/287.1; 435/287.2;
`435/288.3; 435/288.7; 436/164; 436/172;
`436/518; 436/524; 436/527; 436/531; 436/805;
`436/809; 422/58
`Field of Search ......................... 435/6, 287.1, 287.2,
`435/288.3, 288.7; 436/164, 172, 518, 524,
`527, 531, 805, 809; 422/58
`
`References Cited
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`I lllll llllllll Ill lllll lllll lllll lllll lllll 111111111111111111111111111111111
`US006143496A
`[11] Patent Number:
`[45] Date of Patent:
`
`6,143,496
`Nov. 7, 2000
`
`OTHER PUBLICATIONS
`
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`(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
`
`MYR 1015
`Myriad Genetics, Inc. et al. (Petitioners) v. The Johns Hopkins University (Patent Owner)
`IPR For USPN 7,824,889
`
`Page 1 of 30
`
`

`

`6,143,496
`Page 2
`
`OIBER PUBLICATIONS
`
`Cheng et al.; "Analysis of Ligase Chain Reaction Products
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`Kricka et al.; "Imaging of Chemiluminescent Reactions in
`Mesocale Silicon-Glass Microstructures"; J. Biolumin
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`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(cid:173)
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`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'--;.3'Exonuclease Activ(cid:173)
`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 DNA Amplification", Biotech(cid:173)
`niques, vol. 10, No. 1, pp. 76-83 (1991).
`Chang, Physical Chemistry With Applications to Biological
`Systems, 2nd Edition, Sec. 5.4, p. 87.
`Berg, Random Walks in Biology, "Diffusion: Microscopic
`Theory", pp. 10, 49 (1983).
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`Analysis, Proc. Natl. Acad Sci., vol. 93, Genetics, pp.
`5556-5561 (1996).
`Cheng et al.; Chip PCR.11. Investigation of Different PCR
`Amplification Systems in Microfabricated Silicon-Glass
`Chips; Nucleic Acids Research, vol. 24, No. 2, pp. 380-385
`(1996).
`Woolley et al.; "Functional Integration of PCR Amplifica(cid:173)
`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(cid:173)
`side Analogs as Monomers and Incorporated into Oligo(cid:173)
`nucleotides, Analytical Biochemistry, 244, pp. 86-95
`(1997).
`Xu et al., "Direct Measurement of Single-Molecule Diffu(cid:173)
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`vol. 275, pp. 1106-1109 (Feb. 1997).
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`Lide (Editor-In-Chief), p. 6-10 (1993-1994).
`
`Page 2 of 30
`
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`6,143,496
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`Nov. 7, 2000
`Nov. 7, 2000
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`6,143,496
`
`U.S. Patent
`U.S. Patent
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`
`Nov. 7, 2000
`
`Sheet 2 of 7
`
`6,143,496
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`Page 4 of 30
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`Nov. 7, 2000
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`Page 7 of 30
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`

`

`U.S. Patent
`
`Nov. 7, 2000
`
`Sheet 7 of 7
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`6,143,496
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`
`Page 9 of 30
`
`

`

`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(cid:173)
`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(cid:173)
`isms in clinical samples, the genetic identification of foren(cid:173)
`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(cid:173)
`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(cid:173)
`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(cid:173)
`ization of a fluorescent probe or probes to amplified nucleic
`acid. One such assay involves the hybridization of two
`
`10
`
`2
`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
`5 adjacent sites on the target molecule.
`Another assay, which is commercially available, is the
`"TaqMan" fluorescence energy transfer assay and kit, avail(cid:173)
`able from Perkin Elmer, Applied Biosystems Division, Fos(cid:173)
`ter City, Calif. This type of assay is disclosed in the
`publication of Holland et al., Detection of specific Poly(cid:173)
`merase chain reaction product by utilizing the 5'--;.3' 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
`15 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
`20 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(cid:173)
`ized to template ("TaqMan" activity). Nucleolytic degrada-
`25 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-
`30 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(cid:173)
`titation relied on setting up amplification reactions with
`35 known numbers of target nucleic acid molecules and com(cid:173)
`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
`40 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
`45 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
`50 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(cid:173)
`titation by competitive polymerase chain reaction, Proc.
`55 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(cid:173)
`tal nucleic acids can lead to large differences in the amounts
`of their products after the million-fold amplification char-
`60 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
`65 acid, with DNA dyes such as ethidium bromide or SYER TM
`Green I, or "TaqMan" or related fluorescence assays used to
`follow the amount of PCR product accumulated in real time.
`
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`6,143,496
`
`4
`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-
`5 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
`10 acid sequences and/or detection of different sequence vari(cid:173)
`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
`15 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-
`25 cation and detection reactions in parallel on a single speci(cid:173)
`men and for economizing usage of reagents in the process.
`
`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(cid:173)
`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(cid:173)
`fication efficiency in different samples during the exponen(cid:173)
`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 20
`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(cid:173)
`tivity. For example, the "TaqMan" assay requires near
`saturating amounts of PCR product to detect enhanced
`fluorescence. PCRs normally saturate at about 1011 product 30
`molecules/microliter (molecules/µl) due in part to rapid
`reannealing of product strands. To reach this concentration
`of product after 30 cycles in a 10 µl PCR requires at least 103
`starting template molecules (103 x230/10 µl,.,10 11/µl). Some(cid:173)
`what less than this number of starting molecules can be 35
`detected by increasing the number of cycles, and in special
`circumstances even single starting molecules may be detect(cid:173)
`able as described in the publication of Gerard et al., A Rapid
`and Quantitative Assay to Estimate Gene Transfer into
`Retrovirally Transduced Hematopoietic Stem/Progenitor 40
`Cells Using a 96-Well Format PCR and Fluorescent Detec(cid:173)
`tion System Universal for MML V-Based 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 45
`amplicons derived from the primers (so called "primer(cid:173)
`dimers") which interfere with amplification of the desired
`product.
`If the volume of the PCR were reduced 1000-fold to -10
`nanoliters (nl), 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. lx230
`per 10 nanoliters ,.,1011 per microliter. Attempts have been
`made to miniaturize PCR assemblies but no one has devel(cid:173)
`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 60
`to detect single target nucleic acid molecules, miniaturiza(cid:173)
`tion might also facilitate the performance of multiple dif(cid:173)
`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. 65
`For example, it may be desirable to test for the presence of
`multiple different viruses such as HIV-1, HIV-2, HTLV-1,
`
`SUMMARY OF THE INVENTION
`
`According to the present invention, methods and appara-
`tus for performing nucleic acid amplification on a miniatur(cid:173)
`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(cid:173)
`tion also provides an analytical assembly having means to
`determine simultaneously the presence in a sample of mul(cid:173)
`tiple different nucleic acid target molecules.
`According to embodiments of the invention, PCR condi(cid:173)
`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
`50 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(cid:173)
`ments of the invention, sample retaining chambers having
`55 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(cid:173)
`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(cid:173)
`centration of a target nucleic acid molecule in samples of
`about 1 microliter or less. According to some embodiments
`
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`6,143,496
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`15
`
`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
`5 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
`10 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
`20 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
`25 assembly according to an embodiment of the present inven-
`ti on;
`FIG. 8 is a histogram showing the maximum values of the
`fluorescein:rhodamine intensity ratio in over 100 capillary
`reactions of terminally diluted genomic DNA carried out in
`30 an assembly according to the present invention and accord(cid:173)
`ing to a method according to the invention; and
`FIGS. 9-14 are plots of the fluorescein:rhodamine ratio
`along a few representative microcapillaries containing
`35 samples subject to PCR in accordance with the present
`invention.
`
`5
`of the invention, methods are provided for a clinical diag(cid:173)
`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(cid:173)
`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(cid:173)
`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 nl 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 nl or
`less, which allows detection of PCR products generated
`from a single target molecule using the "TaqMan" or other
`fluorescence energy transfer systems.
`According to embodiments of the present invention,
`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 40
`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- 45
`ecules and for quantifying the concentration of a nucleic
`acid molecule in a sample through PCR. The present inven(cid:173)
`tion also provides methods of detecting and quantifying a
`plurality of target DNA sequences.
`The present invention also provides methods and assem(cid:173)
`blies for separating and/or analyzing multiple minute por(cid:173)
`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 55
`portion, and can be used in the fields of fertility,
`immunology, cytology, gas analysis, and pharmaceutical
`screening.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`According to embodiments of the present invention,
`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(cid:173)
`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(cid:173)
`placing the retained sample. In some embodiments, a dis(cid:173)
`placing fluid is used to isolate a retained sample, and the
`50 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(cid:173)
`ration at temperatures of about 95° C. or more, for example,
`at temperatures achieved during thermal cycling under con(cid:173)
`ditions for PCR. The isolated, entrapped or enclosed sample
`60 or portion is preferably protected from contamination and
`evaporation throughout an amplification protocol, for
`exampl