PROVISIONAL PATENT APPLICATION
`
`METHODS AND COMPOSITIONS FOR DETECTING GENETIC MATERIAL
`
`WSGRDocket No. 38938-709.101
`
`Inventors:
`
`Benjamin HINDSON
`Citizen of Australia, Residing at
`1039 Bannock Street
`Livermore, CA 94551
`
`Serge SAXONOV
`Citizen of USA,Residing at
`10 De Anza Ct,
`San Mateo, CA 94402
`
`Philip BELGRADER
`Citizen of USA,Residing at
`89 Robinson Landing Rd.
`Severna Park, MD 21146
`
`Kevin NESS
`Citizen of Canada, Residing at
`24 Baytree Way Apt #10
`San Mateo, CA 94402
`
`Michael LUCERO
`Citizen of USA,Residing at
`634 Pine Terrace
`South San Francisco, CA 94080
`
`Billy COLSTON
`Citizen of USA,Residing at
`9981 Torreon Ave
`San Ramon, CA 94583
`
`Assignee: QuantaLife, Inc.
`7068 Koll Center Pkwy, Ste 401
`Pleasanton, CA 94566
`
`Wek
`Wilson Sonsini Goodrich & Rosati
`PROPESSTONAL CORPORATION
`
`650 Page Mill Road
`Palo Alto, CA 94304
`(650) 493-9300
`(650) 493-6811
`
`ELECTRONICALLY FILED ON: NOVEMBER25,2010
`
`

`

`METHODS AND COMPOSITIONS FOR DETECTING GENETIC MATERIAL
`
`BACKGROUNDOF THE INVENTION
`
`[0001] Fetal aneuploidies are aberrations in chromosome number and commonlyarise as a result of a meiotic
`
`nondisjunction during oogenesis or spermatogenesis; however, certain aneuploidies, such as trisomy 8, result more often
`
`from postzygotic mitotic disjunction (Nicolaidis & Petersen (1998) Human Reproduction 13:313-319). Such aberrations
`
`include both reductions and increases in the normal chromosome numberand can involve autosomesas well as the sex
`
`chromosomes. An example of a reduction aneuploidy is Turner's syndrome, whichis typified by the presence of a single
`
`X sex chromosome. Examples of increases in chromosome number include Down's syndrome(trisomy of chromosome
`
`21), Patau syndrome(trisomy of chromosome 13), Edwards syndrome(trisomy of chromosome 18), and Kleinfelter's
`
`syndrome (an XXY trisomy of the sex chromosomes). Aneuploidies commonly lead to significant physical and
`
`neurological impairments whichresult in a large percentage of affected individuals failing to reach adulthood. In fact,
`
`fetuses having an autosomal aneuploidy involving a chromosomeother than 13, 18, or 21 generally die in utero.
`
`However, certain aneuploidies, such as Kleinfelter's syndrome, present far less pronounced phenotypes and those
`
`affected with other trisomies, such as XXY & XXX,often will matureto be fertile adults. In somecases, partial
`
`aneuploidy resulting in an abnormal copy numberofa portion of a chromosome mayresult from an imbalanced
`
`nondisjunction.
`
`[0002] Prenatal diagnosis of fetal aneuploidies using invasive testing by amniocentesis or Chorionic Villus Sampling
`
`(CVS), are associated with a 0.5% to 2% procedure-related risk of pregnancy loss (D'Alton, M. E., (1994) Semin
`
`Perinatol 18:140-62; Caughey AB (2006) Obstet Gynecol 108:612-6).
`
`[0003] Another barrier to accurately screening fetal aneuploidy is the low concentration of fetal DNA in maternal
`
`plasma, particularly at earlier gestational ages. Single or low multiplex assay approachesare unlikely to provide enough
`
`target counts to differentiate between an aneupoloid fetus (e.g., trisomy of chromosome 21) from a euploid fetus. There
`
`is also, generally, a need in the art for methods and compositions for detecting copy numbervariations in biological
`
`samples, not necessarily from maternal blood.
`
`SUMMARYOF THE INVENTION
`
`[0004] The present disclosure provides methods and compositions for detecting copy numberofa target polynucleotide
`
`within a population of genetic material. Partitioning may be used to subdivide the target polynucleotide into a plurality
`
`of reaction volumes. In somecases, a probe to the target polynucleotide is subdivided into a plurality of reaction
`
`volumes.
`
`
`
`[0005] In somecases, the methods comprise the following steps: a. bindingafirst ligation probe toafirst target
`
`polynucleotide;
`
`b. binding a secondligation probe to a secondtarget polynucleotide; c.
`
`subjecting said first and
`
`secondligation probesto a ligation reaction in order to obtain one or moreligated products; d. partitioning said one or
`
`more ligated products into two or morepartitions; e. amplifying a sequence within said one or moreligated products to
`
`obtain amplified products;
`
`f. determining a numberofsaid partitions that contain said amplified products; and g.
`
`calculating a copy numberofsaidfirst target polynucleotide. In somecases, the target polynucleotideis not partitioned
`
`into said two or morepartitions,
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`[0006] Partitions can include a wide variety of types of partitions, including solid partitions (e.g., wells, tubes, etc.) and
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`fluid partitions (e.g., aqueous droplets within an oil phase, such as a continuousoil phase, or aqueous droplets within a
`
`mixture of at least two immiscible fluids).
`
`Thepartitions mayalso be stable or unstable. For example, in somecases,
`
`during the amplification process said two or morepartitions remain substantially intact. In somecases, the partitions are
`
`aqueous droplets within an oil phase and said aqueous droplets remain substantially intact during the amplification
`
`reaction of the instant methods. Thepartitions (e.g., said aqueous droplets) may also remain substantially intact during
`
`the determination steps, when partitions are evaluated for the presence of one or more target polynucleotides (or probes
`
`to said polynucleotides). The partitions may comprise an amplification reaction that is initiated from said ligated
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`product.
`
`[0007]
`
`Thefirst and secondligation probes may bind(or be designed to bind) a variety of target polynucleotides; often
`
`a first ligation probe bindsa first target polynucleotide and a second ligation probe binds a second polynucleotide. In
`
`somecases, the first and secondligation probes are each designedto bind tosaid first target polynucleotide. In other
`
`cases, said first ligation probe bindsa first target polynucleotide that has a sequencethat differs from the sequence of
`
`said second target poynucleotide.
`
`In somecases, first ligation probe is designed to bind to a polynucleotide sequence
`
`that is conserved between individuals within a species. In somecases, first ligation probe is designed to bind to a
`
`polynucleotide sequence that is conserved across two or more different species. In somecases, a ligation probe binds to
`
`a nonpolymorphic region of a chromosome.
`
`[0008]
`
`In some embodiments, the method comprises ligating multiple ligation probesto saidfirst target polynucleotide.
`
`For example, the method may comprise bindingat least four ligation probesto said first target polynucleotide. In other
`
`cases, at least 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, 20,000,
`
`30,000, 40,000, 50,000, 60,000, 70,000, 100,000, 2,000,000, 3, 000,000, 4,000,000, 5,000,000, 6,000,000, 7,000,000,
`
`8,000,000, 9,000,000 or 10,000,000 ligation probes are used in the methods provided herein. Often, one or moreofsaid
`
`ligation probes bindto a different polynucleotide(e.g., different chromosomes, different regions within the same
`
`chromosome). In somecases, a plurality of first ligation probes(e.g., target ligation probes) are used anda plurality of
`
`second ligation probes(e.g., reference ligation probes) are used in the present methods and compositions. The methods
`
`may further comprise binding at least four ligation probesto said first target polynucleotide andat least four ligation
`
`probes to said second target polynucleotide. In somecases, at least 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, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 100,000, 2,000,000, 3, 000,000,
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`4,000,000, 5,000,000, 6,000,000, 7,000,000, 8,000,000, 9,000,000 or 10,000,000 ligation probes are boundto said first
`
`or said second target polynucleotide.
`
`
`
`[0009] Thefirst ligation probe may bind(or be designedto bind) toafirst region within said first target polynucleotide
`
`and said second ligation probe may bind(or be designed to bind) to a second region within saidfirst target
`
`polynucleotide, wherein said first and second regions do not have identical sequences.
`
`[0010] Often the first target polynucleotide is not identical to said second target polynucleotide. In somecases thefirst
`
`target polynucleotide is identical to the second target polynucleotide.
`
`In some examples,said first target polynucleotide
`
`is a test chromosomeandsaid secondtarget polynucleotide is a reference chromosome. Examplesof test chromosomes
`
`include but are not limited to: chromosome 21, chromosome 13, chromosome 18, and the X chromosome. Said test
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`chromosome mayalso be from the group consisting of chromosome1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
`
`18, 19, 20, 21, 22, X, and Y. A first target polynucleotide may be a segment of a chromosome, such as a segmentof a
`
`chromosomethat is associated with fetal aneuploidy (either the chromosomeor the segment may be associated with fetal
`
`aneuploidy).
`
`[0011] The methods and compositions provided herein often relate to ligating a probe to itself, ligating two probes
`
`together, and/or ligation products of said ligation reactions. Said ligation reactions may result in the ligation of a 5’
`
`region ofsaid first ligation probe to a 3’ region ofsaid first ligation probe to obtain a circular ligated product. In some
`
`cases, a ligation reaction results in the ligation of the 5’ regionofsaid first ligation probe to the 3’ region of said second
`
`ligation probe, in order to obtain a linear ligated product comprising at least a portion ofsaidfirst and secondligation
`
`probes. Said 5’ region andsaid 3’ region of said first ligation probe may each bind (or be designed to bind) adjacent
`
`sequences within said first target polynucleotide. Said adjacent sequences are separated by 0 nucleotides. Said 5’
`
`region and said 3’ regionofsaid first ligation probe may bind, or be designed to bind, neighboring sequences within said
`
`first target polynucleotide. Said neighboring sequences may be separated by at least one nucleotide. In somecases, the
`
`neighboring sequences are separated by a gap ofat least 5, 10, 20, 30, 40 ,50, 100, 200, 300, 400, or 500 nucleotides.
`
`[0012] The ligation reaction may further comprise a template-driven gapfill reaction to incorporate nucleotides in the
`
`gap betweensaid 5’ region and said 3’ regionofsaid first ligation probe (or of said second ligation probe).
`
`[0013] The ligation probes may comprise a site cleavable by an enzyme. For example,the site cleavable by an enzyme
`
`may comprise one or more uracils. The uracils may be separated by other nucleotides in some cases. Thesite cleavable
`
`by an enzyme may comprise a restriction site. The first ligation probe may be of a specific type, such as a molecular
`
`inversion probe, a padlock probe, a linear ligation probe,etc..
`
`[0014] The methods provided herein may further comprise performing an enzymatic reaction to removelinear
`
`polynucleotides or single-stranded polynucleotides or double-stranded polynucleotides. For example, an exonuclease
`
`(e.g., Exo I, II, and/or IID) may be used in the methods described herein. Often, exonuclease treatment removesall, or a
`
`substantial amount, of unboundligation probes from a sample volume.
`
`[0015] The probes provided herein may be conjugated to signaling agent. Saidfirst ligation probe may be conjugated
`
`to a first signaling agent and a secondligation probe is conjugated a second signaling agent. Often, a plurality of such
`
`first and second ligation probes are used in the methods and compositions herein, wherein said probes are conjugated to
`
`the samesignaling agent(e.g., identical fluorophore) or to different signaling agents (e.g., fluorophores ofdifferent
`
`colors). Said first signaling agent may be a fluorescent markerofa first color and said second signaling agent may be a
`
`fluorescent marker of a second color.
`
`[0016] Detection of ligation probesis also often a step in the methods provided herein. The methods may comprise
`
`detecting said first ligation probe withafirst signaling agent and detecting said secondligation probe with a second
`
`signaling agent. Saidfirst ligation probe may comprisea first plurality of ligation probes, wherein each probe within said
`
`plurality is directed to a different region of a first chromosome, and wherein said second ligation probe comprises a
`
`secondplurality of ligation probes, wherein each probe within said plurality is directed to a different region of a second
`
`chromosome. In somecases,said first target polynucleotide is a test chromosome and said second target polynucleotide
`
`is a reference chromosome. In somecases, said first and second ligation probes are conjugated to the samecolor.
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`[0017] The methods and compositions provided herein may also involve a method of detecting copy number of a target
`
`polynucleotide within a population of genetic material comprising: a. bindingafirst ligation probetoafirst target
`
`polynucleotide;
`
`b. binding a secondligation probe to a secondtarget polynucleotide; c.
`
`subjecting said first and
`
`secondligation probesto a ligation reaction in order to obtain one or moreligated products; d. partitioning said one or
`
`more ligated products into two or more aqueous droplets within a continuous oil phase; amplifying a sequence within
`
`said one or more ligated products to obtain amplified products; determining a numberof said two or more aqueous
`
`droplets that contain said amplified products; and g. calculating a copy numberofsaid target polynucleotide based on
`
`said number. In somecases, said target polynucleotide is not partitioned into said two or more aqueous droplets. In some
`
`cases, said target polynucleotide is not amplified.
`
`In some cases, or during said amplifying or determining steps, said
`
`two or more aqueous droplets remain substantially intact.
`
`[0018]
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`In somecases, said two or more aqueous droplets comprise on average more than oneligated probe and said
`
`method further comprises using an algorithm to calculate an average numberoftarget ligated probes per aqueous droplet.
`
`Said two or more aqueous droplets may be greater than 4,000 droplets. In somecases, said two or more aqueous droplets
`
`may be greater than 1,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, or 5,000,000 droplets.
`
`[0019]
`
`In somecases, said droplets are present in a single chamber at a high droplet/ml density. The density may be
`
`greater than 100,000 aqueous droplets/ml. Examples of densities of droplets in a single chamber include: 10,000
`
`droplets/mL, 100,000droplets/mL, 200,000droplets/mL, 300,000droplets/mL, 400,000droplets/mL, 500,000droplets/mL,
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`600,000droplets/mL, 700,000droplets/mL, 800,000droplets/mL, 900,000droplets/mL or 1,000,000droplets/mL. The
`
`droplets used in any of the methods or compositions provided herein may be monodisperse droplets. The droplets may
`
`have, on average, a diameter of between 50 nm and 300 um. In some embodiments, the droplet diameter may be, on
`
`average, about .001, .01, .05, .1, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 100, 120, 130, 140, 150, 160, 180, 200, 300, 400, or
`
`500 microns In somecases, the droplets do not comprise a substantial number of beads conjugated to oligonucleotides.
`
`[0020] The aqueous droplets may be present within an oil fluid or phase. The oil phase may comprise an anionic
`
`flourosurfactant an ammonium salt of an anionic fluorosurfactant, such as KrytoxTM. Krytox may be selected from a
`
`group consisting of Krytox AS, Krytox FSH, and morpholino derivative of Krytox FSH. The oil phase may comprise a
`
`fluorinated oil.
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`[0021] The methods provided herein (e.g., detecting copy number using droplets) can be used to detect said first target
`
`polynucleotide within a population of genetic material comprising less than 1,000 copies of said first target
`
`polynucleotide. In some cases, said two or more aqueous droplets comprise on average more than oneligated probe and
`
`said method further comprises using an algorithm to calculate an average numberoftarget ligated probes per aqueous
`
`droplet.
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`[0022] The droplets may comprisea first target polynucleotide that is a chromosomal segment associated with a
`
`genetic disorder. The droplets may comprise a specific type of ligation probe (e.g., padlock probe, molecular inversion
`
`probe, ligation detection reaction (LDR)probe, etc.). The ligation probe may be subjected to a ligation reaction that
`
`ligates the 5’ region ofthe ligation probe to the 3’ region of the ligation probe.
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`[0023] The methods and compositions provided herein mayalso relate to a method of detecting a fetal genetic
`
`condition comprising: a. obtaining a mixture of maternal and fetal genetic material comprising target polynucleotides; b.
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`combining said mixture with targeting oligonucleotides that bind said target polynucleotides;c. subdividing said targeting
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`oligonucleotides into reaction volumes, wherein at least one of said reaction volumes comprises no target
`
`polynucleotide and no targeting oligonucleotide; d. performing an amplification reaction within said reaction volumes;e.
`
`detecting the presence ofsaid target polynucleotide or said targeting oligonucleotide within said reaction volumes; and
`
`f. determining therelative level of said target polynucleotide in said mixture in order to detect a fetal genetic condition.
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`[0024] The reaction volumes may be aqueous droplets within a continuousoi! phase. The targeting oligonucleotides
`
`may comprise one or more primer pairs;
`
`ligation probes; molecular inversion probes; ligation detection reaction (LDR)
`
`probes; padlock probes; and any combination thereof. The reaction volumes may comprise, on average, greater than one
`
`copy of targeting oligonucleotid, and/or, on average, greater than one copyoftarget polynucleotide. Said reaction
`
`volumes may further comprise primers to a reference polynucleotide. In some cases, said reaction volumes further
`
`comprise a ligation probe to a reference polynucleotide.
`
`[0025]
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`In some embodiments,the ligation probes are amplified within said reaction volumes.
`
`[0026] The fetal genetic material used in the methodsfor detecting a fetal genetic condition may be derived from a
`
`cellular sample that was selectively pre-enriched for fetal genetic material. But, in some embodiments, fetal genetic
`
`material used in the methods for detecting a fetal genetic condition is not derived from a cellular sample that was
`
`selectively pre-enriched for fetal genetic material
`
`[0027]
`
`The target polynucleotide may be within a chromosomeselected from the group consisting of chromosome 18,
`
`13, 21, and X; or from the group consisting of chromosome1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18,
`
`19,20, 21, 22, X, or Y.
`
`[0028]
`
`In somecases, said reaction volumes are aqueous droplets within an oil phase,said targeting oligonucleotides
`
`are ligation probes, and said determining step comprises comparing a numberof droplets comprising an amplified
`
`product of said ligation probes with a number of droplets comprising an amplified product ofligation probes directed to
`
`a reference polynucleotide. In some cases, said reference polynucleotide is a region of a chromosomethatis not
`
`associated with a fetal genetic abnormality.
`
`[0029] As used in the methods and compositions provided herein, said targeting oligonucleotides may be ligation
`
`probes that becomecircular upon ligation following hybridization to a target polynucleotide.
`
`[0030] This disclosure also provides compositions, such as microcapsule compositions, as well as methods for using
`
`said microcapsule compositions. In some cases, the composition is a microcapsule comprising a ligated probe wherein
`
`
`
`said microcapsule is obtained by: a. selectively bindingaplurality of ligation probes to target polynucleotides within a
`
`genetic sample; b.
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`ligating a 5’ end ofat least one of said bound ligation probes to a 3’ end of the sameordifferent
`
`boundligation probe, thereby obtaining at least one ligation product; c.
`
`introducing an aqueoussolution comprising
`
`said at least one ligation product into a device for generating droplets; d. using said device to produce an aqueous
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`droplet comprising said at least one ligation product, wherein said aqueous droplet is within an immiscible fluid; and
`
`e.
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`converting said droplet into a microcapsule comprising a solid-phase exterior. In some cases, said converting
`
`comprises heating above 50 ° C, or heating above 70 ° C. The immiscible liquid (e.g., oil) may comprise a fluorinated
`
`surfactant. In somecases, the aqueous phase comprises a fluorinated surfactant. The oil may be a fluorocarbon oil. The
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`oil phase may comprise an anionic surfactant. The oil phase may comprises ammonium Krytox. In somecases, said
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`microcapsule does not comprise a bead boundto an oligonucleotide. Said microcapsule may remain substantially intact
`
`at temperatures above 70° C. The microcapsule may comprise ligation probes capable of selectively binding toatarget
`
`polynucleotide associated with a genetic disorder. In somecases, said ligation probes are capable of selectively binding
`
`to a target polynucleotide associated with fetal aneuploidy. In somecases, said genetic target is within a chromosome
`
`selected from the group consisting of chromosome 21, chromosome 13, chromosome 18, and the X chromosome. In
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`some cases, the genetic target is within chromosome1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21,
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`22, X, or Y.
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`[0031] The microcapsule may comprise one or moreofsaid ligation probes(e.g., padlock probe, molecular inversion
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`probe, ligation detection reaction (LDR) probe, circular probe, etc.). The microcapsules may comprise a linear probe
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`obtained by linearizing a probe previously circularized after a ligation reaction.
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`In somecases, the microcapsules
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`comprise circularized ligation probe. In somecases, the microcapsules contain linear products ofa ligation detection
`
`reaction (LDR).
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`[0032] The compositions and method provided herein mayalso relate to a water-in-oil mixture comprising two or more
`
`aqueous droplets, wherein at least one of said two or more aqueous droplets comprisesa first ligation probe directed to a
`
`first target polynucleotide and at least one of said two or more aqueous droplets comprises a second ligation probe
`
`directed to a second target polynucleotide. Said first target polynucleotide and said second target polynucleotide may be
`
`the same molecule, or different molecules with identical sequencesor structures, or different molecules with different
`
`sequencesor structures. In somecases, said first target polynucleotide has a different sequence than that of said second
`
`target polynucleotide. In some instances,said first target polynucleotide has an identical sequenceto said secondtarget
`
`polynucleotide. Said first target polynucleotide may comprise a first region within a genomic segmentand said second
`
`target polynucleotide may comprise a second region within said genomic segment, wherein said first region does not
`
`have the same sequence as said second region.
`
`[0033]
`
`In somecases, said water-in-oil mixture further comprises an ammonium krytox surfactant. Said krytox
`
`surfactant may be presentin the oil phase of said mixture at a concentration of at least 0.01%.
`
`[0034]
`
`In somecases, said mixture comprises a ligation probe that is the linearized product of a circular probe that
`
`was subjected to enzymatic cleavage. In somecases, said mixture comprises the circularized probe itself. In some cases,
`
`the ligation probe may comprise an enzymatic cleavagesite, such as where enzymatic cleavage is catalyzed by uracil-N-
`
`glycosylase or a restriction enzyme.
`
`[0035] The present invention includes a methodofdifferential detection of target sequences in a mixture of maternal
`
`and fetal genetic material, comprising the steps of: a) obtaining maternal tissue containing both maternalandfetal
`
`genetic material; b) distributing the genetic material into discrete samples, each sample containing on average not more
`
`than about one target sequence per sample, wherein the discrete sample contains a set of primers to a known target
`
`sequence and/or a set of reference primers to a known reference sequence; c) performing an amplification reaction;
`
`d) detecting the presence of the target or reference sequencein the discrete samples; and e) comparing theratio oftarget
`
`sequences detected to reference sequences detected to determine a differential amount of target sequence. Said method
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`may further comprise a step of comparing theratio of target sequences detected to reference sequences detected to
`
`determinea differential amount of target sequence, wherein a difference in target sequences detected to reference
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`sequences detected indicates a fetal genetic abnormality.
`
`In some embodiments the method is a method of detecting
`
`fetal aneuploidy. In somecases, the target sequence is a marker for aneuploidy and the reference sequenceis diploid in
`
`maternal and fetal genetic material. The maternal tissue may be maternal peripheral blood, blood plasma or serum, or
`
`other tissue described herein. In some embodiments, the reaction samples are in aqueous phases in an emulsion. In some
`
`cases, detecting the presence of the target or reference sequence further includes hybridizing it in situ with a nucleic acid
`
`having a fluorescent label. In some cases, the number of reaction samplesis at least about 10,000. In somecases, steps
`
`b) to e) are repeated with a primer set to a different target sequence. In somecases, the reaction volume comprises more
`
`than one primerset with each primer set to a particular target sequence. In somecases, the reaction volume comprises
`
`more than one reference primer set with each primer set to a particular reference sequence. Examples of primer sets that
`
`can be used include primer sets specific for human chromosome 21, human chromosome 18, human chromosome 13, or
`
`human chromosome X.
`
`In some cases, aneuploidy is detected wherethe ratio of target to reference sequence detected
`
`is greater than 1. In somecases, an aneuploidy is detected wherethe ratio oftarget to reference sequence detected is less
`
`than 1. In somecases, the target sequenceis at least a portion of a CFTR, Factor VIII (F8 gene), beta globin,
`
`hemachromatosis, G6PD, neurofibromatosis, GAPDH,beta amyloid, or pyruvate kinase gene.
`
`INCORPORATION BY REFERENCE
`
`[0036] All publications and patent applications mentioned in this specification are herein incorporated by reference in
`
`their entirety and to the same extent as if each individual publication or patent application was specifically and
`
`individually indicated to be incorporated by reference.
`
`RELATED APPLICATIONS
`
`[0001] This application relates to U.S. Provisional Application No. 61/264,591, filed on November 25, 2009; U.S.
`
`Provisional Application No. 61/309,837, filed on March 2, 2010; U.S. Provisional Application No. 61/309,845,filed on
`
`March 2, 2010; U.S. Provisional Application No. 61/317,635, filed on March 25, 2010; U.S. Provisional Application No.
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`61/317,639, filed on March 25, 2010; U.S. Provisional Application No. 61/317,684, filed on March 25, 2010; U.S.
`
`Provisional Application No. 61/341,065, filed on March 25, 2010; U.S. Provisional Application No. 61/341,218, filed on
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`March 25, 2010; U.S. Provisional Application No. 61/380,981, filed on September 8, 2010; U.S. Provisional Application
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`No. 61/409,106, filed on November 1, 2010; U.S. Provisional Application No. 61/409,473, filed on November 2, 2010;
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`and U.S. Provisional Application No. 61/410,769, filed on November 5, 2010, each of which is incorporated herein by
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`referencein its entirety.
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`BRIEF DESCRIPTION OF THE FIGURES
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`[0037] The novel features of the invention are set forth with particularity in the appended claims. A better
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`understanding of the features and advantages of the present invention will be obtained by referenceto the following
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`detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the
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`accompanying figures of which:
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`[0038] FIG. 1 is a schematic overview illustrating the steps that can be taken to detect copy numbervariations in a
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`patient sample through the use of droplet digital PCR andligation probes.
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`[0039]
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`FIG. 2 is a schematicillustration of an example of steps that can be followed to detect changes in the number of
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`chromosomes(or portions thereof) in a sample.
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`[0040] FIG. 3 depicts a workflow of an exemplary method for diagnosing fetal aneuploidy.
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`[0041] FIG. 41s a schematic illustration of the use of Molecular Inversion Probes (MIPs) to detect two genetic targets.
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`[0042] FIG. 5 shows multiplexing of the MIP approachto increase sensitivity of detection of genetic targets.
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`[0043] FIG. 6 shows a two-color system for detection of nucleic acids in droplets using universal primers and universal
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`probes without cleavage.
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`[0044] FIG. 7 shows a schemefor detecting two genetic targets with two colors using a ligation-detection reaction
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`(LDR) followed by PCR in droplets.
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`[0045] FIG. 8 depicts the use of multiplexed oligonucleotides for LDR-PCR in droplets to enhancesensitivity of
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`detection.
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`[0046] FIG. 9 depicts a computer useful for displaying, storing, retrieving, or calculating data or results obtained by the
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`methods and compositions described herein.
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`[0047] FIG. 10 showsa correlation between numberof input copies of template DNA and numberofpositive droplets
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`(or counts), and increased sensitivity when using 12-plex MIPs (lower panels) compared to 3-plex MIPs (upper panels),
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`for a test sample.
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`[0048] FIG. 11 shows numberofpositive droplets (or counts) versus number of template copies, for a reference
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`sample.
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`[0049] FIG. 12 showshybridization efficiency for different template copy numbers andat different levels of MIP
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`multiplexing.
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`[0050] FIG. 13 shows 24 MIPsdirected to different regions within Chromosome | (SEQ ID NOS:1-24).
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`[0051] FIG. 14 shows 24 MIPsdirected to different regions within Chromosome 21 (SEQ ID NOS: 25-48).
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`[0052] FIG. 15 showsa 3-plex set of MIPs directed to different regions within Chromosome 1 (SEQ ID NOS: 49-51)
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`and a 12-plex set of MIPs directed to different regions within Chromosome | (SEQ ID NOS: 52-63).
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`[0053] FIG. 16 showsa 3-plex set of MIPs directed to different regions within Chromosome 21 (SEQ ID NOS: 64-66)
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`and a 12-plex set of MIPs directed to different regions within Chromosome 21 (SEQ ID NOS: 67-78).
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`[0054] FIG. 17 shows exemplary universal primers and probes for the detection of a MIP (SEQ ID NOS: 79-82).
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`General Overview
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`DETAILED DESCRIPTION OF THE INVENTION
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`[0055] This disclosure provides methods and compositions for detecting genetic variations in a biological sample. In
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`somecases, this disclosure provides methods and compositions for detecting the numberofcopies of a target
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`polynucleotide (e.g., chromosome, chromosomefragment, gene, etc.) within a biological sample. In some cases, methods
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`and compositions for detecting genetic mutations and/or single nucleotide polymorphisms (SNPs) within a biological
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`sampleare also provided.
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`[0056] This disclosure also provides compositions and methodsfor detecting fetal aneuploidy, or other genetic
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`abnormality, in a biological sample derived from maternal tissue. Often such a biological sample comprises a mixture of
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`maternal and fetal nucleic acids (e.g., DNA, RNA). Aneuploidy is a chromosomal abnormality, and refers to an
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`aberration in the copy number of a chromosome, or fragmentthereof, or portion thereof. The methods and materials
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`described herein apply techniques for analyzing numerous nucleic acids contained in a tissue sample, such as blood
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`(whole blood or peripheral blood), serum or plasma, containing a mixture of DNA (and/or DNA fragments) from both
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`the mother andthe fetus, and allowing detection of small differences between target and reference DNA levels that may
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`indicate fetal aneuploidy.
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`[0057] As used herein, copy numbervariations (CNVs)refer to gains or losses of segments of genetic material. There
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`are large numbers of CNV regions in humansand a broad rangeof genetic diversity among the general population.
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`CNVsalso play a role in many human genetic disorders. The methodis espec