PROVISIONAL PATENT APPLICATION
`
`METHODS AND COMPOSITIONS FOR DETERMINING NUCLEIC ACID FRAGMENTATION
`
`WSGRDocket No. 38938-718.101
`
`Inventor(s):
`
`Serge SAXONOV,
`Citizen of USA, Residing at
`10 De Anza Ct.,
`San Mateo, CA 94402
`
`Benjamin HINDSON,
`Citizen of Australia, Residing at
`1039 BannockStreet
`Livermore, CA 94551
`
`Bill COLSTON,
`Citizen of USA, Residing at
`9981 Torreon Avenue
`San Ramon, CA 94583
`
`WR
`Wilson Sonsini Goodrich & Rosati
`PROFESSIONAL CORPORATION
`
`650 Page Mill Road
`Palo Alto, CA 94304
`(650) 493-9300 (Main)
`(650) 493-6811 (Facsimile)
`
`Filed Electronically on: April 15, 2010
`
`WSGRDocket No. 38938-718.101
`
`

`

`METHODS AND COMPOSITIONS FOR DETERMINING NUCLEIC ACID FRAGMENTATION
`
`BACKGROUNDOF THE INVENTION
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`[0001] Measuring whether two or more genomicloci that are genetically linked(i.e. on the same
`
`chromosome, same plasmid,etc.) are still linked or separated from one another in a given sample can provide
`
`useful informationforlife sciences applications, the medicalfield, and in applied markets, such as forensics.
`
`There is a need for improved methods and compositions for measuring whether two or more genomiclocithat
`
`are genetically linked arestill linked or are separated from one anotherin a given sample.
`
`SUMMARYOF THE INVENTION
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`In general, in one aspect, a method for determining the probability of fragmentation of two
`[9002]
`genetically linked loci in a nucleic acid sample is provided comprising: a. performing digital PCR (€PCR) on
`
`said sample, wherein the dPCR comprises separating the nucleic acid into separated units; b. determining a
`
`first sum of the number of units with signal indicating the presence ofa first locus and the numberof units
`
`with signal indicating the presenceofthe first locus and a second locus; c. determining a second sum ofthe
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`numberof units with signal indicating the presence of the second locus and the numberof units with signal
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`indicating the presence ofthe first locus and the second locus; and d. inputing the first and second sumsinto
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`an algorithm to determine the percentage of the two genetically linked loci in the sample that are fragmented.
`
`INCORPORATION BY REFERENCE
`
`[0003] All publications, patents, and patent applications mentioned in this specification are herein
`
`incorporated by reference to the same extent as if each individual publication, patent, or patent application
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`wasspecifically and individually indicated to be incorporated by reference.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
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`[0004] The novel features of the invention are set forth with particularity in the appended claims. A better
`
`understanding of the features and advantages of the present invention will be obtained by reference to the
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`following detailed description that sets forth illustrative embodiments, in which the principles of the invention
`
`are utilized, and the accompanying drawings of which:
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`[0005] Figure 1 illustrates FAM and VIC signals on a nucleic acid separated by 1K, 10K, or 100K bases.
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`DETAILED DESCRIPTION OF THE INVENTION
`[0006] Provided herein are methods, compositions, and kits for determining whether two or more genetic
`loci that are genetically linked (i.e. on the same nucleic acid molecule, chromosome, same plasmid,etc.) are
`
`still linked or separated from one another in a given sample. The methods provided herein can be applied to
`
`samples used in a polymerase chain reaction (PCR), e.g., a digital PCR (dPCR). The digital PCR can be
`droplet digital PCR.
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`[0007] For example, using droplet digital PCR (ddPCR), a duplex reaction can be performedtargeting two
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`genomicloci, e.g., two genes on a common chromosome. The droplets can be categorized into four
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`populations according to their fluorescence. For example, if a FAM-labeled probe is used to detect to one
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`loci, and a VIC-labeled probe is used to detect anotherloci, the four populations can be FAM+/VIC4+,
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`FAM+/VIC-, FAM-/VIC+, and FAM-/VIC-. By comparing the numberof droplets with each of these
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`populations, it is possible to determine the frequency at which loci co-segregate to the same droplet. Using
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`Poissonstatistics, the percentage of species that are actually linked to one another can be estimated versus
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`instances where two separated loci are in the same droplet by chance.
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`[0008] The numberof genetically linked loci that can be examined to determineif they arestill linked in a
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`sample or are separated in the sample using the methods, compositions, and kits described herein can be
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`about, at least about, or more than about2, 3, 4, 5, 6, 7, 8, 9, or 10. The numberof genetically linked loci that
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`can be examinedto determineif they arestill linked in a sample or are separated in the sample using the
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`methods, compositions, and kits described herein can be about 2-10, 2-8, 2-6, 2-4, 3-10, 3-8, 3-6, 4-10, or 4-6.
`
`The numberofbase pairs between each ofthe genetically linked loci can be about, at least about, or less than
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`about 10 bp, 25 bp, 50bp, 75 bp, 100 bp, 250 bp, 500 bp, 750 bp, 1000 bp, 2000 bp, 3000 bp, 4000 bp, 5000
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`bp, 6000 bp, 7000 bp, 8000 bp, 9000 bp, 10,000 bp, 15,000 bp, 20,000 bp, 33,000 bp, 50,000 bp, 75,000 bp,
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`100,000 bp, 250,000 bp, 500,000 bp, 750,000 bp, 1,000,000 bp, 1,250,000 bp, 1,500,000 bp, 2,000,000 bp,
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`5,000,000 bp, or 10,000,000 bp. The numberof base pairs between each of the genetically linked loci can be
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`about 10-10,000,000 bp, 100-10,000,000 bp, 1,000-10,000,000 bp, 1,000-1,000,000 bp, 1,000-500,000 bp,
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`1,000-100,000 bp, 3000-100,000 bp, 1000-33,000 bp, 1,000-10,000 bp, or 3,000-33,000 bp.
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`[0009] Oneofthe loci genetically linked to another locus can be a commonreference, e.g., RPP30. Any
`
`genetically linked loci can be used in the methods described herein.
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`[0010] A label (Fluorophore, dye) used on a probe (e.g., a Taqman probe) to detect a locus in the methods
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`described herein can be, e.g., 6-carboxyfluorescein (FAM), tetrachlorofluorescin (TET), 4,7,2'-trichloro-7’-
`
`phenyl-6-carboxyfluorescein (VIC), HEX, Cy3, Cy 3.5, Cy 5, Cy 5.5, Cy 7, tetramethylrhodamine, ROX, and
`
`JOE. Thelabel can be an Alexa Fluordye, e.g., Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594,
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`633, 647, 660, 680, 700, and 750. The label can be Cascade Blue, Marina Blue, Oregon Green 500, Oregon
`
`Green 514, Oregon Green 488, Oregon Green 488-X, Pacific Blue, Rhodamine Green, Rhodol Green,
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`Rhodamine Green-X, Rhodamine Red-X, and Texas Red-X. A unique label can be used to detect each
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`different locus in an experiment.
`
`[0011] A sample comprising genetically linked loci can be subjected to fragmentation before being analyzed
`
`by the methods, compositions, or kits described herein. A sample comprising genetically linked loci can be
`
`fragmented by, e.g., mechanical shearing, passing the sample through a syringe, sonication, heat treatment
`
`(e.g., 30 mins at 90°C), and/or nuclease treatment (e.g., with DNase, RNase, endonuclease, exonuclease,
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`restriction enzyme). A sample comprising genetically linked loci can be subjected to no or limited processing
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`before being analyzed.
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`[0012] Applications
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`[0013] Fragmentation/linkage assays described herein can be usedas a diagnostic.
`[0014] Determining whether nucleic acids are linked or separated (fragmented) can provide useful
`
`information for a variety of applications. Determining whether nucleic acids are linked or separated
`
`(fragmented) can be used to study apoptosis. Apoptosis is a process of programmedcell death. Signs of
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`apoptosis can include,e.g., blebbing, loss of cell membrane asymmetry and attachment,cell shrinkage,
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`nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation. During apoptosis,
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`endogenenous endonucleases can cleave chromatin DNAinto internucleosomal fragments of about 180-200
`
`basepairs or multiples of about 180-200 base pairs. Using the methods, compositions, and/or kits described
`
`herein, the fragmentation status of different forms of genetic material at different stages of the apoptosis can
`
`be determined. The methods described herein can be used with one or more other methodsto analyze
`
`apopotosis, including, e.g., flow cytometry, fluorescent assays, or (TUNEL) (terminal deoxynucleotidyl
`
`transferase dUTP nick end labeling).
`
`[0015] Determining whethernucleic acids are linked or separated (fragmented) can be used to study a
`
`polymerase(e.g., DNA polymerase, RNA polymerase, reverse transcriptase). For example, the processivity
`
`of a polymerase can be determined(e.g., to determine the percentage of nascent strandsthat are full length
`
`versus partial length, one can measure how manytruncated versions of a gene are present by counting the
`
`numberoffirst half copies versus last half copies of a gene). Because synthesis occurs 5’ to 3’, it is expected
`that more ofthe 1st half (5’ end) would be produced than the last half (3’ end).
`[0016] Determining whetherlociare linked or separated (fragmented) in a sample can be useful for studying
`
`one or morerestriction enzymes, RNAzymes, DNAzymes, exonucleases, endonucleases, RNases, DNase,
`
`etc., to determine the efficiency of cleavage (i.e. separation to two linked targets).
`
`[0017] Determining whether genetic loci are linked or separated (fragmented) can be useful for studying
`
`RNAsplicing, genetic rearrangement, localization of genes, and DNA rearrangementin cancer.
`
`[0018] Determining the degree of degradation (fragmentation) of forensic genetic material can help
`
`determine what analyses can be successfully performed prior to wasting precious sample. Determining
`
`whether nucleic acidsare linked or separated (fragmented) can be useful for determining an expected defect
`
`from perfect integer value copy numberestimates due to random shearing of the DNA.
`
`[0019] Targetpolynucleotide
`
`[0020]
`
`In one aspect, the methods described herein can be used for detecting one or more target nucleic acid
`
`molecules. The term polynucleotide, or grammatical equivalents,typically refer to at least two nucleotides
`covalently linked together. A nucleic acid described herein will generally contain phosphodiester bonds,
`although in somecases, as outlined below (for example in the construction ofprimers and probes such as
`label probes), nucleic acid analogs are included that may have alternate backbones, comprising, for example,
`phosphoramide (Beaucageetal., Tetrahedron 49(10):1925 (1993) and references therein; Letsinger, J. Org.
`
`Chem. 35:3800 (1970); Sprinzlet al., Eur. J. Biochem. 81:579 (1977); Letsingeret al., Nucl. Acids Res.
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`14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsingeret al., J. Am. Chem. Soc. 110:4470 (1988);
`
`and Pauwelset al., Chemica Scripta 26:141 91986)), phosphorothioate (Maget al., Nucleic Acids Res.
`
`19:1437 (1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al., J. Am. Chem. Soc. 111:2321
`
`(1989), O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical
`
`Approach, Oxford University Press), and peptide nucleic acid (also referred to herein as “PNA’”) backbones
`and linkages (see Egholm, J. Am. Chem. Soc. 114:1895 (1992); Meieret al., Chem.Int. Ed. Engl. 31:1008
`(1992); Nielsen, Nature, 365:566 (1993); Carlssonet al., Nature 380:207 (1996), all of which are incorporated
`
`by reference). Other analog nucleic acids include those with bicyclic structures including locked nucleic acids
`
`(also referred to herein as “LNA”), Koshkin et al., J. Am. Chem. Soc. 120.13252 3 (1998); positive
`
`backbones (Denpcyet al., Proc. Natl. Acad. Sci. USA 92:6097 (1995); non-ionic backbones (U.S. Pat. Nos.
`
`5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem.Intl. Ed. English
`
`30:423 (1991); Letsingeret al., J. Am. Chem. Soc. 110:4470 (1988); Letsingeret al., Nucleoside &
`
`Nucleotide 13:1597 (1994); Chapters 2 and 3, ASC Symposium Series 580, “Carbohydrate Modifications in
`
`Antisense Research”, Ed. Y. 8. Sanghui and P. Dan Cook; Mesmaekeret al., Bioorganic & Medicinal Chem.
`
`Lett. 4:395 (1994); Jeffs et al., J. Biomolecular NMR 34:17 (1994); TetrahedronLett. 37:743 (1996)) and
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`non-ribose backbones, including those described in U.S. Pat. Nos. 5.235,033 and 5,034,506, and Chapters 6
`
`and 7, ASC Symposium Series 580, “Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghui
`
`and P. Dan Cook. Nucleic acids containing one or more carbocyclic sugarsare also included within the
`definition of nucleic acids (see Jenkinset al., Chem. Soc. Rev. (1995) pp 169 176). Several nucleic acid
`
`analogs are described in Rawls, C & E NewsJun. 2, 1997 page 35. “Locked nucleic acids” are also included
`
`within the definition of nucleic acid analogs. LNAsare a class of nucleic acid analogues in whichthe ribose
`
`ring is “locked” by a methylene bridge connecting the 2’-0 atom with the 4’-C atom. All of these references
`
`are hereby expressly incorporated by reference. These modifications of the ribose-phosphate backbone can be
`
`done to increase the stability and half-life of such molecules in physiological environments. For example,
`
`PNA:DNAand LNA-DNAhybrids can exhibit higher stability and thus can be used in some embodiments.
`
`The target nucleic acids can be single stranded or double stranded, as specified, or contain portions of both
`
`double stranded or single stranded sequence. Depending on the application, the nucleic acids can be DNA
`
`(including, e.g., genomic DNA, mitochondrial DNA, and cDNA), RNA(including, e.g., mRNA and rRNA)
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`or a hybrid, where the nucleic acid contains any combination of deoxyribo- and ribo-nucleotides, and any
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`combination of bases, including uracil, adenine, thymine, cytosine; guanine, inosine, xathanine
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`hypoxathanine, isocytosine, isoguanine, etc.
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`[0021] The methods and compositions provided herein can be used to evaluate a quantity of polynucleotides
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`(e.g., DNA, RNA, mitochondrial DNA, genomic DNA, mRNA,siRNA, miRNA, cRNA,single-stranded
`
`DNA,double-stranded DNA,single-stranded RNA, double-stranded RNA, tRNA, rRNA, cDNA,etc.). The
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`methods and compositions can be used to evaluate a quantity of a first polynucleotide comparedto the
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`quantity of a second polynucleotide. The methods can be used to analyze the quantity of synthetic plasmids
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`in a solution; to detect a pathogenic organism (e.g., microbe, bacteria, virus, parasite, retrovirus, lentivirus,
`HIV-1, HIV-2, influenza virus, etc.) within a sample obtained from a subject or obtained from an
`environment. The methods also can be used in other applications wherein a rare population of polynucleotides
`
`exists within a larger population of polynucleotides.
`
`[0022] Sample Acquisition and Preparation
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`[0023] This starting material can be obtained in some cases fromahospital, laboratory, clinical or medical
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`laboratory. The sample is taken from a subject. The sample can comprise nucleic acid, e.g., RNA or DNA.
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`The sample can comprisecell-free nucleic acid.
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`In somecases, the sample is obtained from a swab ofa
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`surface, such as a door or benchtop.
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`[0024] Droplet digital PCR.
`
`[0025]
`
`In somecases,less than 0.00001, 0.00005, 0.00010, 0.00050, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2,
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`' 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, or 10 copies of target polynucleotide are detected. In somecases,less than 1,
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`2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,
`150, 200, 250, 300, 350, 400, 450, or 500 copies ofa target polynucleotide are detected.. In somecases, the
`droplets described herein are generated at a rate of greater than 1, 2, 3, 4, 5, 10, 50, 100, 200, 300, 400, 500,
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`600, 700, 800, 900, or 1000 droplets/second.
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`[0026] An integrated, rapid, flow-through thermal cycler device can be used in the methods of the provided
`
`invention. See, e.g., International Application No. PCT/US2009/005317, filed 9-23-2009. In such an
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`integrated device, a capillary is wound around a cyclinder that maintains 2, 3, or 4 temperature zones. As
`droplets flow throughthe capillary, they are subjected to different temperature zones to achieve thermal
`cycling. The small volumeof each droplet results in an extremely fast temperaturetransition as the droplet
`
`enters each temperature zone.
`
`[0027] A digital PCR device(e.g., droplet digital PCR device) for use with the methods, compositions, and
`
`kits described herein can detect multiple signals (see e.g. US Provisional Patent Application No. 61/454,373,
`
`filed March 18, 2011, herein incorporated by referencein its entirety).
`
`[0028] Amplification
`
`[0029] Techniques for amplification of target and reference sequences are knownin theart, and include the
`
`methods described in US Patent No. 7,048,481. Briefly, the techniques include methods and compositions
`
`that separate samples into small droplets, in some instances with each containing on averageless than one
`
`nucleic acid molecule per droplet, amplifying the nucleic acid sequence in each droplet and detecting the
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`presenceofa particular target sequence. In somecases, the sequencethat is amplified is present on a probe to
`
`the genomic DNA,rather than the genomic DNAitself.
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`[0030] Primers are designed according to known parameters for avoiding secondary structures andself-
`
`hybridization. In some embodiments, different primer pairs will anneal and melt at about the same
`
`temperatures, for example, within 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10°C of another primerpair. In somecases,
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`greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 500, 1000, 5000, 10,000 or
`more probesare initially used. Such probes maybeable to hybridizeto the genetic targets described herein.
`
`[0031] Primers can be prepared by a variety of methodsincluding but not limited to cloning of appropriate
`
`sequencesanddirect chemical synthesis using methods well knownin the art (Naranget al., Methods
`
`Enzymol. 68:90 (1979); Brownet al., Methods Enzymol. 68:109 (1979)). Primers can also be obtained from
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`commercial sources such as Operon Technologies, Amersham Pharmacia Biotech, Sigma, and Life
`
`Technologies. The primers can have an identical melting temperature. The lengths of the primers can be
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`extended or shortenedat the 5' end orthe 3' end to produce primers with desired melting temperatures. In a
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`preferred embodiment, one of the primers of the primepair is longer than the other primer. In a preferred
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`embodiment, the 3' annealing lengths of the primers, within a primerpair, differ. Also, the annealing position
`
`of each primerpair can be designed such that the sequence and length of the primerpairs yield the desired
`
`melting temperature. The simplest equation for determining the melting temperature of primers smaller than
`
`25 basepairs is the Wallace Rule (Td=2(A+T)+4(G+C)). Computer programscan also be used to design
`
`primers, including but notlimited to Array Designer Software (Arrayit Inc.), Oligonucleotide Probe Sequence
`
`Design Software for Genetic Analysis (Olympus Optical Co.), NetPrimer, and DNAsis from Hitachi Software
`
`Engineering. The TM (melting or annealing temperature) of each primeris calculated using software
`
`programs such as Net Primer (free web based program at http://premierbio soft.
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`com/netprimer/netprlaunch/netprlaunch.html; internet address as of Apr. 17, 2002). In another embodiment,
`
`the annealing temperature of the primers can be recalculated and increased after any cycle of amplification,
`
`including but not limited to cycle 1, 2, 3, 4, 5, cycles 6-10, cycles 10-15, cycles 15-20, cycles 20-25, cycles
`
`25-30, cycles 30-35, or cycles 35-40. After the initial cycles of amplification, the 5' half of the primersis
`
`incorporated into the products from eachloci of interest, thus the TM can be recalculated based on both the
`
`sequencesof the 5' half and the 3' half of each primer.
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`[0032] The annealing temperature of the primers can be recalculated and increased after any cycle of
`
`amplification, including but not limited to cycle 1, 2, 3, 4, 5, cycles 6-10, cycles 10-15, cycles 15-20, cycles
`
`20-25, cycles 25-30, cycles 30-35, or cycles 35-40. After the initial cycles of amplification, the 5' half of the
`
`primersis incorporated into the products from each loci of interest, thus the TM can be recalculated based on
`
`both the sequencesofthe 5' half and the 3' half of each primer. Any DNA polymerasethat catalyzes primer
`
`extension can be used includingbut not limited to E. coli DNA polymerase, Klenow fragment of E. coli DNA
`polymerase 1, T7 DNA polymerase, T4 DNA polymerase, Taq polymerase, Pfu DNA polymerase, Vent DNA
`
`polymerase, bacteriophage 29, REDTaq™. Genomic DNA polymerase, or sequenase. Preferably, a
`
`thermostable DNA polymeraseis used. A hot start PCR can also be performed wherein the reaction is heated
`
`to 95°C. for two minutes prior to addition of the polymerase or the polymerase can be kept inactive until the
`first heating step in cycle 1. Hot start PCR can be used to minimize nonspecific amplification. Any number of
`PCRcycles can be used to amplify the DNA,including butnot limited to 2, 5, 10, 15, 20, 25, 30, 35, 40, or 45
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`cycles.
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`[0033] Amplification of target nucleic acids can be performed by any means knownin theart. In somecases,
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`target nucleic acids are amplified by polymerase chain reaction (PCR). Examples of PCR techniques that can
`
`be used include, but are not limited to, quantitative PCR, quantitative fluorescent PCR (QF-PCR), multiplex
`
`fluorescent PCR (MF-PCR), real time PCR(RT-PCR), single cell PCR,restriction fragment length
`
`polymorphism PCR (PCR-RFLP), PCR-RFLP/RT-PCR-RFLP,hot start PCR, nested PCR,in situ polonony
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`PCR,insitu rolling circle amplification (RCA), bridge PCR,picotiter PCR and emulsion PCR. Othersuitable
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`amplification methods include the ligase chain reaction (LCR), transcription amplification, molecular
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`inversion probe (MIP) PCR,self-sustained sequencereplication, selective amplification of target
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`polynucleotide sequences, consensus sequence primed polymerase chain reaction (CP-PCR),arbitrarily
`
`primed polymerase chain reaction (AP-PCR), degenerate oligonucleotide-primed PCR (DOP-PCR)and
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`nucleic acid based sequence amplification (NABSA). Other amplification methods that can be used herein
`
`include those described in U.S. Pat. Nos. 5,242,794; 5,494,810; 4,988,617; and 6,582,938. In some
`
`embodiments, amplification of target nucleic acids may occur ona bead. In other embodiments, amplification
`
`does not occur on a bead.
`
`[0034]
`
`In some cases, thermocycling reactions are performed on samples contained in droplets. In some
`
`preferred cases, the droplets remain intact during thermocycling. Droplets may remain intact during
`
`thermocycling at densities of greater than about 10,000 droplets/mL, 100,000 droplets/mL, 200,000
`
`droplets/mL, 300,000 droplets/mL, 400,000 droplets/mL, 500,000 droplets/mL, 600,000 droplets/mL,
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`700,000 droplets/mL, 800,000 droplets/mL, 900,000 droplets/mL or 1,000,000 droplets/mL. In other cases,
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`two or more droplets may coalesce during thermocycling. In other cases, greater than 100 or greater than
`
`1,000 droplets may coalesce during thermocycling.
`
`[0035] Universal probes may be designed by methods knownin the art. In some embodiments, the probeis a
`
`random sequence. The universal probe may beselected to ensure that it does not bind the target
`
`polynucleotide in an assay, or to other non-target polynucleotides likely to be in a sample (e.g., genomic DNA
`
`outside the region occupied by the target polynucleotide).
`
`[0036] Fluorescence detection can be achieved using a variety of detector devices equipped with a module to
`
`generate excitation light that can be absorbedbya fluorescer, as well as a moduleto detect light emitted by
`
`the fluorescer. In somecases, samples (such as droplets) may be detected in bulk. For example, samples may
`
`be allocated in plastic tubes that are placed in a detector that measures bulk fluorescence from plastic tubes.
`
`In some cases, one or more samples (such as droplets) may be partitioned into one or more wellsofa plate,
`
`such as a 96-well or 384-well plate, and fluorescence of individual wells may be detected using a fluorescence
`
`plate reader.
`
`[0037]
`
`In somecases, the detector further comprises handling capabilities for droplet samples, with
`
`individual droplets entering the detector, undergoing detection, and then exiting the detector. For example, a
`
`flow cytometry device can be adapted for use in detecting fluorescence from droplet samples. In somecases, a
`
`microfluidic device equipped with pumpsto control droplet movementis used to detect fluorescence from
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`droplets in single file. In somecases, droplets are arrayed on a two-dimensional surface and a detector moves
`relative to the surface, detecting fluorescence at each position containing a single droplet.
`[0038] Following acquisition of fluorescence detection data, a computer is used in some cases to store and
`process the data. A computer-executable logic may be employed to perform such functionsas subtraction of
`
`backgroundfluorescence, assignmentof target and/or reference sequences, and quantification of the data. A
`
`computer may be useful for displaying, storing, retrieving, or calculating diagnostic results from the
`
`molecular profiling; displaying, storing, retrieving, or calculating raw data from genomic or nucleic acid
`
`expression analysis; or displaying, storing, retrieving, or calculating any sample or patient information useful
`in the methods ofthe present invention.
`[0039] Following digital PCR of samples having primers to amplify a target and a reference sequence,the
`
`numberofpositive samples having a target sequence and the numberofpositive samples having a reference
`sequence can be compared.
`
`[0040] Droplet Generation
`
`[0041] The present disclosure includes compositions and methods using droplet digital PCR. The droplets
`
`described herein include emulsion compositions (or mixtures of two or more immiscible fluids) described in
`
`US Patent No. 7,622,280, and droplets generated by devices described in International Application No.
`
`PCT/US2009/005317, filed 9-23-2009, first inventor: Colston. The term emulsion, as used herein, refers to a
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`mixture of immiscible liquids (such as oil and water). Oil-phase and/or water-in-oil emulsions allow for the
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`compartmentalization of reaction mixtures within aqueousdroplets. In preferred embodiments, the emulsions
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`comprise aqueous droplets within a continuousoil phase. In other cases, the emulsions provided herein are
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`oil-in-water emulsions, wherein the droplets are oil droplets within a continuous aqueous phase. The droplets
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`provided herein are designed to prevent mixing between compartments, with each compartmentprotecting its
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`contents from evaporation and coalescing with the contents of other compartments.
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`[0042] The mixtures or emulsions described herein may bestable or unstable. In preferred embodiments, the
`emulsionsarerelatively stable and have minimal coalescence. Coalescence occurs whensmall droplets
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`combine to form progressively larger ones. In somecases, less than 0.00001 %, .00005%,
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`.00010%,.00050%,.001%, , .005%, .01%, .05%, .1%, .5%, 1%, 2%. 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 7%,
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`8%, 9%, or 10% of droplets generated from a droplet generator coalesce with other droplets. The emulsions
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`may also have limited flocculation, a process by which the dispersed phase comesout of suspension in flakes.
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`[0043] Splitting a sample into small reaction volumesas described herein, may enable the use of reduced
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`amounts of reagents, thereby lowering the material cost of the analysis. Reducing sample complexity by
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`partitioning also improves the dynamic range of detection, since higher-abundance molecules are separated
`from low-abundance moleculesin different compartments, thereby allowing lower-abundance molecules
`greater proportional access to reaction reagents, which in turn enhancesthe detection of lower-abundance
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`molecules.
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`In somecases, droplets may be generated having an average diameter of about .001, .01, .05, .1, 1,5,
`[0044]
`10, 20, 30, 40, 50, 60, 70, 80, 100, 120, 130, 140, 150, 160, 180, 200, 300, 400, or 500 microns. Microfluidic
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`methods of producing emulsion droplets using microchannel cross-flow focusing or physical agitation are
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`knownto produce either monodisperse or polydisperse emulsions. In some embodiments, the droplets are
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`monodisperse droplets. In somecases, the droplets are generated such that the size of said droplets does not
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`vary by more than plus or minus 5% ofthe average size of said droplets. In some cases, the droplets are
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`generated such that the size of said droplets does not vary by more than plus or minus 2% ofthe average size
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`of said droplets. In somecases, a droplet generator will generate a population of droplets from a single
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`sample, wherein none ofthe droplets vary in size by more than plus or minus .1%, .5%, 1%, 1.5%, 2%, 2.5%,
`3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% of the average size of the
`total population of droplets.
`[0045] Higher mechanicalstability is useful for microfluidic manipulations and higher-shear fluidic
`processing (e.g. in microfluidic capillaries or through 90 degree turns, such as valves, in fluidic path). Pre-
`and post- thermally treated droplets or capsules are mechanically stable to standard pipet manipulations and
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`centrifugation.
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`[0046]
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`In somecases, the droplet is formed by flowing an oil phase through an aqueous sample. In some
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`preferred embodiments, the aqueous phase comprises a buffered solution and reagents for performing a PCR
`reaction, including nucleotides, primers, probe(s) for fluorescent detection, template nucleic acids, DNA
`polymerase enzyme, andoptionally, reverse transcriptase enzyme.
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`{0047]
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`In somecases, the aqueous phase comprises a buffered solution and reagents for performing a PCR
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`reaction without solid-state beads, such as magnetic-beads. In somecases, the buffered solution may
`comprise about 1, 5, 10, 15, 20, 30, 50, 100, or 200 mM Tris. In somecases, the concentration of potassium
`chloride may be about 10, 20, 30, 40, 50, 60, 80, 100, 200 mM. In one preferred embodiment, the buffered
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`solution comprises 15 mM Tris and 50 mM KCI. In somecases, the nucleotides comprise
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`deoxyribonucleotide triphosphate molecules, including dATP, dCTP, dGTP, dTTP, in concentrations of about
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`50, 100, 200, 300, 400, 500, 600, or 700 uM each. In some cases dUTPis added within the aqueous phase to
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`a concentration of about 50, 100, 200, 300, 400, 500, 600, or 700, 800, 900, or 1000 1M. In somecases,
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`magnesium chloride (MgCl2) is added to the aqueous phaseat a concentration of about 1.0, 2.0, 3.0, 4.0, or
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`5.0 mM. In one preferred embodiment, the concentration of MgCl2 is 3.2mM.
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`[0048] A non-specific blocking agent such as BSA orgelatin from bovine skin may be used, wherein the
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`gelatin or BSAis present in a concentration range of approximately 0.1-0.9% w/v. Other possible blocking
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`agents may include betalactoglobulin, casein, dry milk, or other commonblocking agents. In somecases,
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`preferred concentrations of BSA and gelatin are 0.1% w/v.
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`[0049] Primers for amplification within the aqueous phase may have a concentration of about 0.1, 0.2, 0.3,
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`0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 uM. In one preferred embodiment, the concentration of primers is 0.5 uM.
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`In somecases, the aqueous phase comprises one or moreprobesfor fluorescent detection, at a concentration
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`of about 0.1, 0.2, 0.3, 0.4, or 05 uM. In one preferred embodiment, the concentration ofprobes for
`fluorescent detection is 0.25 uM. Amenable ranges for target nucleic acid concentrations in PCR are between
`about 1 pg and about 500 ng.
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`[0050]
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`In some embodiments, the aqueous phase mayalso comprise additives including, but not limitedto,
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`non-specific background/blocking nucleic acids (e.g., salmon sperm DNA), biopreservatives (e.g. sodium
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`azide), PCR enhancers(e.g. Betaine, Trehalose, etc.), and inhibitors (e.g. RNAse inhibitors).
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`[0051]
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`In somecases, a non-ionic Ethylene Oxide/Propylene Oxide block copolymeris added to the aqueous
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`phase in a concentration of about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1.0%.
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`Common biosurfactants include non-ionic surfactants such as Pluronic F-68, Tetronics, Zonyl FSN.In one
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`preferred embodiment, Pluronic F-68 is present at a concentration of 0.5% w/v.
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`[0052]
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`In some cases magnesium sulfate may be substituted for magnesium chloride, at similar
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`concentrations. A wide range of common, commercial PCR buffers from varied ven