(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(19) World Intellectual Property
`Organization
`International Bureau
`
`(10) International Publication Number
`(43) International Publication Date
`WO 2013/101896 Al
`WIPO! PCT
`4 July 2013 (04.07.2013)
`
`
`Ss
`
`G1)
`
`International Patent Classification:
`C120 1/8 (2006.01)
`
`Qy)
`
`International Application Number:
`
`PCT/US2012/071758
`
`@2)
`
`International Filing Date:
`
`(25)
`
`Filing Language:
`
`(26)
`
`Publication Language:
`
`27 December 2012 (27.12.2012)
`
`English
`
`English
`
`US
`
`(30)
`
`(71)
`
`(72)
`
`(74)
`
`(81)
`
`Priority Data:
`61/582,200
`30 December 2011 (30.12.2011)
`
`Applicant: QUEST DIAGNOSTICS INVESTMENTS
`INCORPORATED [US/US];
`300 Delaware Avenue,
`Wilmington, Delaware 19899 (US).
`
`Inventor: STROM, Charles M.; 2939 Calle Gaucho, San
`Clemente, California 92673 (US).
`
`Agents: SCHORR, Kristel et al.; Foley & Lardner LLP,
`3000 K Street NW, Suite 600, Washington, District of
`Columbia 20007 (US).
`
`Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY,
`BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM,
`DO, DZ, EC, EE, EG, ES, FL GB, GD, GE, GH, GM, GT,
`
`HN, HR, HU,ID,IL,IN,IS, JP, KE, KG, KM, KN, KP,
`KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD,
`ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NL
`NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU,
`RW, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TI,
`TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA,
`ZM, ZW.
`
`(84)
`
`Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, RW,SD, SL, SZ, TZ,
`UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ,
`TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,
`EE, ES, FL FR, GB, GR, HR, HU,IE, IS, IT, LT, LU, LV,
`MC, MK, MT, NL, NO,PL, PT, RO, RS, SE, SL, SK, SM,
`TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW,
`ML, MR, NE, SN, TD, TG).
`Declarations under Rule 4.17:
`
`as to applicant's entitlement to apply for and be granted a
`patent (Rule 4.17(ii))
`
`as to the applicant's entitlement to claim the priority of the
`earlier application (Rule 4.17(iii))
`Published:
`
`with international search report (Art. 21(3))
`
`(64) Title: NUCLEIC ACID ANALYSIS USING EMULSION PCR
`
`(57) Abstract: The present invention provides methods for analyzing large nucleic acids including chromosomes and chromosomal
`fragments. In one aspect, the present invention provides a method of nucleic acid analysis comprising the stcps of (a) obtaining a
`sample of nucleic acid comprising at least one chromosomeor fragment greater than about 1 000 base pairs in length and containing
`a target region; (b) creating an cmulsion in which cachdrop of the cmulsion contains an average of between about 0-2, 0-1.75, 0-1.5,
`Q-1.0, 0-0.75, 0-0.5, or fewer chromosomesor fragments of step (a), (c) performing emulsion PCR, (d) quantifying the numberof
`emulsion droplets containing amplified nucleic acid fromthe target region; (e) calculating the ratio of droplets containing amplified
`mucleic acid from the target region to total droplets; and (f) comparingthe ratio of step (e) to a reference ratio representing a known
`genotype.
`
`
`
`
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`wo2013/101896At|INTININMTIATIANATAAA
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`NUCLEIC ACID ANALYSIS USING EMULSION PCR
`
`CROSS REFERENCE TO RELATED APPLICATIONS
`
`[0001] This application claims the benefit under 35 U.S.C. § 119(e) of United States
`
`Provisional Application Serial Number 61/582,200, filed December 30, 2011, which is
`
`incorporated by referencein its entirety.
`
`FIELD OF THE INVENTION
`
`[0002] The present invention relates to methods of nucleic acid analysis, in particular
`
`methods using emulsion PCR in analysis of large nucleic acid fragments or whole
`
`chromosomes.
`
`BACKGROUNDOF THE INVENTION
`
`[0003] The following discussion of the background of the invention is merely provided to
`
`aid the reader in understanding the invention and is not admitted to describe or constitute
`
`prior art to the present invention.
`
`[0004] Many modern advancesin cellular and molecular biology are rooted in the advent of
`
`large-scale amplification of nucleic acids and analytical methods dependent thereon. A
`
`number of methods are knownin the art for performing such amplification of template
`
`nucleic acid molecules to generate populations of substantially identical copies. One
`
`technique that is particularly amenable to high throughput applications is emulsion
`
`polymerasechain reaction (“emulsion PCR” or “emPCR’’).
`
`[0005] Emulsion PCRis performedby isolation of individual DNA molecules along with
`
`primer-coated beads in aqueousdroplets within an oil phase. A PCR step coats each bead
`
`with clonal copics of the DNA molecule which are then immobilized for later sequencing.
`
`Emulsion PCRis used in a number of commercial methods, such as those of 454 Life
`
`Sciences, and SOLID sequencing, (developed by Agencourt, now Applied Biosystems).
`
`Current emulsion PCR techniques involve the use of small fragments of DNA, which renders
`
`it unsuitable for analysis of certain genotypes such as those depending onanallelic linkage,
`
`and other applications for which assessment of large nucleic acids are required. Therefore, a
`
`need remains for emulsion PCR-based analytical methods that may applied to the evaluation
`
`of large nucleic acids including, for example, for monosomalanalysis.
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`SUMMARYOF THE INVENTION
`
`[0006] The present invention is based on the discovery that certain nucleic acid preparation
`
`techniques, such as those used in molecular combing, will facilitate the use of large nucleic
`
`acid fragments in emulsion PCR and subsequentanalysis.
`
`[0007]
`
`In one aspect, the present invention provides a method of nucleic acid analysis
`
`comprising the steps of (a) obtaining a sample of nucleic acid comprising at least one
`
`chromosomeor fragment greater than about 1000 base pairs in length and containing a target
`
`region; (b) creating an emulsion in which each drop of the emulsion contains an average of
`
`between about 0-2, 0-1.75, 0-1.5, 0-1.0, 0-0.75, 0-0.5, or fewer chromosomesor fragments of
`
`step (a), (c) performing emulsion PCR,(d) quantifying the number of emulsion droplets
`
`containing amplified nucleic acid from the target region; (e) calculating the ratio of droplets
`
`containing amplified nucleic acid from the target region to total droplets; and (f) comparing
`
`the ratio of step (e) to a reference ratio representing a known genotype.
`
`[0008]
`
`In another aspect, the present invention provides a method for determining the
`
`genotype of a subject (e.g., human) suspected to carry a 2+0 genotype comprising the steps of
`
`(a) obtaining a DNA sample comprising the locus of interest from the subject, (b) creating an
`
`emulsion in which each drop of the emulsion contains an average of between about 0-2, 0-
`
`1.75, 0-1.5, 0-1.0, 0-0.75, 0-0.5, or fewer chromosomesor fragments of step (a), (c)
`
`performing emulsion PCR,(d) quantifying the number of emulsion droplets containing
`
`amplified nucleic acid from the locus; (e) comparing the number of emulsion droplets from
`
`step (d) with the number of emulsion droplets containing amplified nuclcic acid from the
`
`locus of a control 1+1 genotype sample, wherein a 1+1 genotypic sample will show
`
`successful amplification in about two times the emulsion droplets as the control and a
`
`negative sample with no successful amplification will indicate that the subject has a deletion
`
`of both alleles. In some embodiments, the locus of interest is the SMN1 gene on human
`
`chromosome5.
`
`[0009] A “2+0 genotype” ofa diploid cell, as used herein, refers to a duplication of a
`
`genetic sequence on a chromosome with a deletion or other types of disruption of the
`
`sequence at the counterpart chromosome. The wild-type of a 2+0 genotype can be referred to
`
`as a “1+1 genotype” in which each chromosomeof a pair contains a copy of the sequence. A
`
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`2+0 genotype can result from improper genetic recombination or translocation between the
`
`chromosomes, withoutlimitation.
`
`[0010]
`
`Since a 2+0 genotype and the corresponding 1+1 genotype have the same number
`
`of copies of the genetic sequence in a cell, they cannot be distinguished by dosageanalysis
`
`(e.g., quantitative PCR with the entire cell) alone. The present disclosure, however, provides
`
`a ready solution. This is because the chromosome fragment enclosed in each emulsion drop
`
`is large enough to include both copies of the duplication in a 2+0 genotype, whereas, for a
`
`1+1 genotype, the two copies that are located on separated on different chromosomes would
`
`be separated into different drops.
`
`[0011]
`
`The methods of the present disclosure can also be used to improve sequencing
`
`efficiency experimentally and/or computationally. It is contemplated that the inclusion of a
`
`large (e.g., longer than 1000 basepairs, 10 kilobases (kb), 100 kb, 200 kb, 300 kb, 400 kb,
`
`500 kb, 600 kb, 700 kb, 800 kb, 900 kb or 1,000 kb) intact fragment of a chromosome(a)
`
`enables subsequent sequencing ofa relative longer sequence, and (b) facilitates sequence
`
`alignment among sequences obtained from within a drop since no sequence overlap is
`
`expected from sequencing a single copy of a genetic sequence.
`
`[0012]
`
`In some embodiments, the nucleic acid of step (a) is processed by (i) obtaining a
`
`sample of nucleic acid in a solution (e.g., an aqueoussolution); (ii) retracting the meniscus of
`
`the solution; and (iii) immobilizing the ends of the nucleic acid to facilitate isolation of the
`
`individual nucleic acid molecules. In some embodiments, the nucleic acids are immobilized
`
`by allowing the cnds to bind to ionisable groups on a solid substrate (c.g, a silated glass
`
`plate). Immobilization preferably is performed at a pH below the pKa of the ionizable groups
`
`of the solution. In some embodiments, the rate of retraction of the meniscus is constant rate,
`
`and be about 300 um/sec.
`
`[0013]
`
`In other embodiments, the emulsion may be created by mechanical agitation or
`
`microfluidic droplet generation. The droplets may cach be between about 15 and about 100
`
`pL in volume. Further, the at least one chromosomeor fragment may be greater than 10
`
`kilobases (kb),. greater than 100 kilobases (kb), greater than 200 kb, greater than 300 kb,
`
`greater than 400 kb, greater than 500 kb, greater than 600 kb, greater than 700 kb, greater
`
`than 800 kb, greater than 900 kb, greater than 1,000 kb or more in length. In further
`
`embodiments, the chromosome or fragment is between about 200 and about 700 kb in length.
`
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`[0014] As used herein, the term “stretching” refers to any process by which nucleic acid
`
`molecules in solution are elongated (i.e., unwound). In one embodiment, stretching is
`
`performed using the force of a receding meniscus to produces a high-density array of nucleic
`
`acid molecules. In other embodiments, the nucleic acids are at least about 10 kilobases (kb),
`
`100 kb, 200 kb, 300 kb, 400 kb, 500 kb, 600 kb, 700 kb, 800 kb, 900 kb, 1,000 kb or more in
`
`length. In certain embodiments, the nucleic acids are between about 200 and 700 kilobases
`
`(kb) in length. In other embodiments, the nucleic acids subject to stretching are chromosomes
`
`or chromosome fragments.
`
`[0015] As used herein, the term “emulsion drop” or “emulsion droplet” refers to any primer-
`
`coated aqueous droplet contained in an oil solution. The emulsion drop may be between
`
`about 15 and about 100 pL in volume, and may contain a nucleic acid template.
`
`[0016] As used herein, the term “carrier state’’ is meant a person in which only one
`
`chromosomeof a chromosomepair encodesa functional copy of the gene of interest. The
`
`copy of the gene of interest which is non-functional may be non-functional as a result of an
`
`inactivating mutation (1.c., the gene maybe present but inactive) or may bepartially or totally
`
`deleted from chromosome(e.g., resulting from a chromosomaldeletion or translocation). In
`
`the case of the SMNI gene located on chromosome5, a carricr state occurs when onepair
`
`member of chromosome 5 lacks an SMN|1 locus, but two copies of the gene are translocated
`
`onto the second pair member. This mutation produceslittle or no phenotypic effect when
`
`present in a heterozygous condition with a non-diseaserelated allele, but produces a “disease
`
`state” when a person is homozygous, 1.e., both pair members of chromosome5 lack
`
`functional SMN1 nucleic acid sequences.
`
`[0017] By “primer” is meant a sequence of nucleic acid, preferably DNA, that hybridizes to
`
`a substantially complementary target sequence andis recognized by DNA polymerase and
`
`serves as a substrate to initiate DNA replication in an amplification reaction (e.g., PCR).
`
`[0018] As used hercin, the term “substantially complementary” is meant that two sequences
`
`hybridize under stringent hybridization conditions. The skilled artisan will understandthat
`
`substantially complementary sequences need not hybridize along their entire length. In
`
`particular, substantially complementary sequences comprise a contiguous sequence of bases
`
`that do not hybridize to a target sequence, positioned 3' or 5' to a contiguous sequence of
`
`bases that hybridize under stringent hybridization conditions to a target sequence.
`
`4.
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`[0019] As used herein, “amplification” is meant one or more methods knownintheart for
`
`copying a target nucleic acid, thereby increasing the numberof copies of a selected nucleic
`
`acid sequence. Amplification may be exponential or linear. A target nucleic acid may be
`
`either DNA or RNA. The sequences amplified in this manner form an “amplicon.”
`
`[0020] As usedherein, “biological sample” is meant a sample obtained from a biological
`
`source. When obtained from a subject (e.g., a human patient), a biological sample may, by
`
`way of non-limiting example, consist of or comprise blood, serum, plasma, cerebrospinal
`
`fluid (CSF), urine, feces, tissue samples including biopsy samples(e.g., obtained by a fine
`
`needle aspirate (FNA)), and those obtained by non-invasive techniques such as epidermal
`
`samples(e.g., cheek swabs), amniotic fluid, bone marrow sample and/or chorionic villi. The
`
`term biological sample includes samples which have been processed to release or otherwise
`
`make available a nucleic acid for detection as described herein. For example, a biological
`
`sample may include a cDNAthat has been obtained by reverse transcription of RNA from
`
`cells in a biological sample.
`
`[0021] By "anchoring" of the macromolecule on the surface, there should be understood an
`
`attachmentresulting from a chemical reactivity both through a covalent linkage and a
`
`noncovalent linkage such as a linkage resulting from physicochemicalintcractions, such as
`
`adsorption.
`
`DETAILED DESCRIPTION
`
`[0022] The present invention is directed to the methods of nucleic acid analysis and, in
`
`particular, analysis of large nucleic acids using emulsion PCR techniques. The invention is
`
`particularly useful in situations wherein a genotype 1s characterized by anallelic linkage or
`
`lack thereof, and identification or diagnosis requires methods of analysis that keep the linkage
`
`intact. In the presently disclosed methods, whole chromosomesorlarge fragments arc
`
`pretreated to facilitate their cmulsion into droplcts prior to undergoing cmulsion PCR.
`
`Nucleic Acid Preparation
`
`[0023] As discussed below in greater detail, standard emulsion PCR is conducted using
`
`small nucleic acid fragments. In order to properly quantify many samples, however,it is
`
`imperative that larger fragments, or even whole chromosomes, be placed inside each droplet.
`
`In the present methods, the samples are therefore subjected to preparation steps prior to
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`emulsion PCR to ensure that a large fragment size is maintained. In preferred methods, the
`
`preparation is similar to that undergone in molecular combing assay.
`
`[0024]
`
`In molecular combing, deproteinised DNA molecules in solution with a random-coil
`
`conformation attach with non-sequence specificity to a silanised hydrophobic glass surface
`
`by their extremities. While not wishing to be bound by any theory,it is understood that pH-
`
`induced denaturation of the DNA ends exposes the hydrophobic domainsof the bases,
`
`allowing a strong interaction with the hydrophobic surface. The glass surface is mechanically
`
`pulled out of the solution at a constant speed (300 um/sec) where the receding air-water
`
`meniscus exerts a constant perpendicular force on the attached DNA molecules. This
`
`constant perpendicular force is central to obtaining uniformly stretched DNA of a singular
`
`orientation.
`
`[0025] Other techniquescan also result in the stretching and the alignment of molecules. A
`
`dynamic orientation of molecules in solution, anchored at one end, can be obtained by, for
`
`example, electrophoresis or by a hydraulic flow.
`
`[0026] The force of the receding meniscusis insufficient to break either the DNA
`
`extremity-surface interaction or covalent bonds within the DNA molecule; however, the
`
`receding meniscusexerts sufficient force to overstretch DNA from its random-coil
`
`conformation to 150 percent of its molecular contour length. This degree of extension
`
`corresponds to a 65 pN applied force determined by DNA force/extension curves. Once in
`
`contact with air, the DNA sticks onto the surface preventing molecule retraction. DNA is
`
`most likely attached to the silanised surface at several closcly spaced intervals, as determined
`
`by examining recoil following DNA photo-cleavage.
`
`[0027] This anchorage of the macromolecule can be achieved directly on (or with) the
`
`surface, or indirectly, that is to say via a linkage such as another molcculc, cspecially another
`
`molecule with biological activity. When the anchorage is achicved indirectly, the
`
`macromolecule can be grafted chemically on the said linkage, or can interact
`
`physicochemically with the said linkage, in particular when the said intermediate linkageis a
`
`molecule with biological activity recognizing and interacting with the said macromolecule.
`
`Thus, in orderto carry out the direct or indirect anchoring of the macromolecule on the
`
`surface, it is possible to use a solid surface having certain specificities. It is in particular
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`possible to use certain pretreated surfaces which makeit possible to attach certain proteins or
`
`DNA, whether modified or otherwise.
`
`[0028]
`
`Suitable surfaces for anchoring large DNA fragments and/or chromosomesare
`
`commercially available (Covalink, Costar, Estapor, Bangs, Dynal for example) in various
`
`forms having at their surface COOH, NH2 or OH groupsfor example. It is, in this case,
`
`possible to functionalize the DNA with a reactive group, for example an amine, and carry out
`
`a reaction with these surfaces. However, these methods require specific functionalization of
`
`the DNAto beattached. A technique allowing anchorage without prior treatment of the DNA
`
`has also been described. This process consists in causing a free phosphate at the 5' end of the
`
`DNA molecule to react with a secondary amine of the surface (NH Covalink surface).
`
`Anchoring by adsorption can be achieved by adsorption of the end of the molecule by
`
`controlling the surface charge by means of the pH, the ionic content of the medium or the
`
`application of an electric voltage over the surface given the differences in adsorption between
`
`the ends of the molecule and its middle part. According to the present invention,
`
`nonfunctionalized DNA molecules were thus anchored, by way of cxample, on surfaces
`
`coated with molecules cnding with a vinyl or amine group such as polylysine molecules, or
`
`various surfaces such as glass, coated with silanc type molecules ending with vinyl or aminc
`
`groups oralternatively glass cover slips previously cleaned in an acid bath.In this latter case,
`
`the surface of the glass indeed has SiOH groups.
`
`[0029]
`
`In all these cases, the pH range where the DNA 1s anchored is chosen to be between
`
`a state of complete adsorption and an absence of adsorption,the latter being situated at a
`
`more basic pH.It is understood that this technique is very general and can be extended by
`
`persons skilled in the art to numerous types of surfaces. It is also possible to functionalize the
`
`DNAwith a first reactive group or a protein with a first binding pair memberin order to
`
`cause it to react with a surface coated with a second reactive group or with a second binding
`
`pair member which is capable of reacting specifically with cach other. The binding pair
`
`members maybea pair of the type: biotin/streptavidin (Zimmerrann and Cox) or
`
`digoxigenin/antibody directed against digoxigenin (anti-DIG) for example (Smith et al.,
`
`Science 258, 1122 (1992)).
`
`[0030]
`
`Preferably, the anchoring surfaces will have a low fluorescence level so as not to
`
`interfere with the detection of the molecules after their alignment, in particularif the
`
`detection is done by fluorescence. The support can therefore have a surface coated with a
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`reactive group or with a molecule with biological activity. By "affinity", there should be
`
`understood here both a chemical reactivity and an adsorption of any type, this under optional
`
`conditions of attachment of the molecules onto the exposed group, modified or otherwise. In
`
`one embodiment, the surface is essentially compact, that is to say that it limits access by the
`
`macromolecule with biological activity to the inner layers and/or to the support, this in order
`
`to minimize nonspecific interactions. It is also possible to use surfaces coated with a reactive
`
`exposed group (for example NH> , COOH, OH, CHO)or with a macromolecule with
`
`biological activity (for example: proteins, such as streptavidin or antibodies, nucleic acids
`
`such as oligonucleotides, lipids, poly-saccharides and derivatives thereof) which is capable of
`
`attaching an optionally modified part of the molecule. Thus, surfaces coated with
`
`streptavidin or with an antibody according to known processes ("Chemistry of Protcin
`
`Conjugation and Cross-linking", S.C. Wong, CRC Press (1991)) are capable of attaching a
`
`macromolecule having, at a specific sitc, a biotin or an antigen. Likewisc, surfaccs treated so
`
`as to have single-stranded oligonucleotides can serve in order to anchor on them DNAsor
`
`RNAshaving a complementary sequence.
`
`[0031] Among the surfaces having an exposed reactive group, there may be mentioned
`
`those on which the exposed group is a -COOH, -CHO, NH2, -OH group, or a vinyl group
`
`containing a double bond -CH-CH) whichis usedasit is or which can be activated so as to
`
`give especially -CHO, -COOH, -NHz2 or OH groups. The supports with highly specific
`
`surfaces accordingto the present invention can be obtained using various processes. There
`
`may be mentioned by way of example: (A) a layer of carbon-containing, optionally branched,
`
`polymerat least 1 nm thick, having reactive groups as defined above and (B) surfaces
`
`obtained by depositing or anchoring on a solid support one or more molecular layers; the
`
`latter can be obtained by forming successive layers attached through noncovalent linkages, as
`
`non-limiting example, Langmuir-Blodgett films, or by molecular self assembly, this allowing
`
`the formation of a layer attached by covalent linkage. In the first case, the surface can be
`
`obtained by polymerization of at least one monomergenerating at the surface of the polymer
`
`the said exposed group,or alternatively by partial depolymerization of the surface of a
`
`polymer to generate the said exposed group, or alternatively by deposition of polymer. In
`
`this process, the polymer formed has vinyl linkages such as a polyene derivative, especially
`
`surfaces of the synthetic rubber type, such as polybutadiene, polyisoprene or natural rubber.
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`[0032]
`
`In the second case, the highly specific surface contains: on a support, a substantially
`
`monomolecular layer of an organic compound ofelongated structure havingat least: an
`
`attachment group havingan affinity for the support, and an exposed group having noorlittle
`
`affinity for the said support and the said attachment group under attachment conditions, but
`
`optionally having, after chemical modification following the attachment, an affinity for one
`
`type of biological molecule. The attachment canfirst of all be of the noncovalent type,
`
`especially of the hydrophiliclhydrophilic and hydrophobic/ hydrophobictype, as in
`
`Langmuir-Blodgett films (K. B. Blodgett, J. Am. Chem. Soc. 57, 1007 (1935). In this case,
`
`the exposed group or the attachment group will be either hydrophilic or hydrophobic,
`
`especially alkyl or haloalkyl groups such as CH3 , CF; , CHF3 , CH? F, the other group being
`
`hydrophilic. The attachment can also be of the covalent type, the attachment group will, in
`
`this case, react chemically with the support. Certain surfaces of similar structure have
`
`already been mentionedin the electronic ficld, especially when the attachments are covalent,
`
`L. Netzer and J. Sagiv, J. Am. Chem. Soc. 105, 674 (1983) and U.S. Pat. No. 4,539,061.
`
`Amongthe attachment groups, there must be mentioned more particularly the groups ofthe
`
`metal alkoxide or semiconductor type, for example silane, especially chlorosilane, silanol,
`
`methoxy- and ethoxysilane, silazane, as well as phosphate, hydroxyl, hydrazide, hydrazine,
`
`amine, amide, diazonium, pyridine, sulfate, sulfonic, carboxylic, boronic, halogen, acid
`
`halide, aldehyde groups.
`
`[0033] The combing process produces a high-density array of DNA moleculesthat are
`
`between 200 and 700 kilobases (kb) in length. DNAfibers are uniformly stretched along
`
`their length regardless of sequence content. This uniform stretching provides a length scale
`
`relating physical distance on the surface to genomic length, i.e.
`
`1 m= 2 kb. Typically, 50-
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`100 diploid human genomesare combed onto 22 x 22 mm slides, making quantitative studies
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`possible from the detection of multiple events per assay. For a complete discussion of
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`molecular combing procedures, see, e.g., U.S. Patent No. 6,548,255 to Bensimonet al., which
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`is herein incorporated by reference in its entirety; see also Lebofsky and Bensimon, Single
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`DNA Molecule Analysis, Briefings in Functional Genomics and Proteomics, Vol. 1, No. 4,
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`383-396 (2003).
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`Recovery and Dilution of Combed Nucleic Acid Products
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`[0034] The combed nucleic acids are then recovered. Recovery may be achieved by any
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`means knownin the art, such as, for example, the addition of a solution containing an agent,
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`e.g., a restriction endonuclease or a component with a pH above the pKaof the ionized
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`groups, that acts to release the anchorto the silated glass, or, in the alternative, recovery may
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`be achieved physically by blotting the slide with NA-45 paper, washingit in TE buffer,
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`heating in an elution buffer, and extracting using phenol and ethanol followed by
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`centrifugation.
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`Emulsion PCR
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`[0035]
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`The present methodsutilize emulsion PCR for amplification of a monosomeor
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`fragment of interest. Typical embodiments of emulsion PCR methods includecreating a
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`stable emulsion of two immiscible liquids to create liquid (e.g., aqueous) droplets within
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`whichreactions may occur. In particular, the aqueous droplets of an emulsion amenable for
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`use in PCR methods mayincludea first fluid, such as a water based fluid suspended or
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`dispersed as droplets (also referred to as a discontinuous phase) within another fluid, such as
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`a hydrophobic fluid (also referred to as a continuous phase) that typically includesan oil.
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`Examples ofoil that may be employed include, but are not limited to, mineraloils, silicone
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`basedoils, or fluorinated oils.
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`[0036] Optionally, in some embodiments, the emulsion may include one or more
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`surfactants that act to stabilize the emulsion, which maybeparticularly useful for specific
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`processing methods such as PCR. Some embodiments of surfactant may include one or more
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`of a silicone or fluorinated surfactant. For example, one or more non-ionic surfactants may be
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`employedthat include, but are not limited to, sorbitan monooleate (also referred to as Span™
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`80), polyoxycthylcnesorbitsan monoolcate (also referred to as Tween™ 80), or in some
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`preferred embodiments, dimethicone copolyol (also referred to as Abil® EM90),
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`polysiloxane, polyalkyl polyether copolymer, polyglycerol esters, poloxamers, and
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`PVP/hexadecane copolymers (also referred to as Unimer U-151), or in morepreferred
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`embodiments, a high molecular weightsilicone polyether in cyclopentasiloxane (also referred
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`to as DC 5225C available from Dow Corning).
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`[0037] The aqueous droplets may range in size depending on the composition of the
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`emulsion components or composition, contents contained therein, and formation technique
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`employed. The described emulsions create the microenvironments within which chemical
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`reactions, such as PCR, may be performed. For example, template nucleic acids andall
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`reagents necessary to perform a desired PCR reaction may be encapsulated and chemically
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`isolated in the droplets of an emulsion. Additional surfactants or other stabilizing agents may
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`be employed in some embodiments to promote additional stability of the droplets as
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`described above. Thermocycling operations typical of PCR methods may be executed using
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`the droplets to amplify an encapsulated nucleic acid template resulting in the generation of a
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`population comprising many substantially identical copies of the template nucleic acid. In
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`some embodiments, the population within the droplet may be referred to as a "clonally
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`isolated", "compartmentalized", "sequestered", "encapsulated", or "localized" population.
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`Also in the present example, someorall of the described droplets may further encapsulate a
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`solid substrate such as a bead for attachment of template and amplified copies of the
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`template, amplified copies complementary to the template, or combination thereof. Further,
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`the solid substrate may be cnabled for attachment of other type of nucleic acids, reagents,
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`labels, or other molecules of interest.
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`[0038] Embodiments of an emulsion useful with the presently described invention may
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`include a very high density of droplets or microcapsules enabling the described chemical
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`reactions to be performed in a massively parallel way. Additional cxamples of cmulsions
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`employed for amplification and their uses for sequencing applications are described in U.S.
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`Pat. Nos. 7,638,276; 7,622,280; and U.S. patent application Scr. Nos. 10/767,899; and
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`11/045,678, each of which is hereby incorporated by reference herein in its entirety for all
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`purposes.
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`[0039] Also embodiments sometimes referred to as UltraDeep Sequencing, generate target
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`specific amplicons for sequencing may be cmployed with the presently described invention
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`that include using sets of specific nucleic acid primers to amplify a selected target region or
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`regions from a sample comprising the target nucleic acid. Further, the sample mayinclude a
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`population of nucleic acid molecules that are knownor suspected to contain sequence
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`variants comprising sequence composition associated with a research or diagnostic utility
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`where the primers may be employed to amplify and provide insight into the distribution of
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`sequence variants in the sample. For example, a method for identifying a sequence variant by
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`specific amplification and sequencing of multiple alleles in a nucleic acid sample may be
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`performed. The nucleic acid is first subjected to amplification by a pair of PCR primers
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`designed to amplify a region surroundingthe region of interest or segment commonto the
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`nucleic acid population. Each of the products of the PCRreaction (first amplicons) is
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`subsequently further amplified individually in separate reaction vessels such as an emulsion
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`based vessel described above. The resulting amplicons(referred to herein as second
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`amplicons), each derived from one memberofthe first population of amplicons, are
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`sequenced andthe collection of sequences are used to determinean allelic frequency of one
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`or more variants present. Importantly, the method does not require previous knowledge ofthe
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`variants present and can typically identify variants present at <1% frequencyin the
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`population of nucleic acid molecules.
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`[0040]
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`Some advantages of the described target specific amplification and sequencing
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`methods include a higherlevel of sensitivity than previously achi