(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
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`(19) World Intellectual Property Organization _'
`International Bureau
`'
`
`(43) International Publication Date
`16 August 2007 (16.08.2007)
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` (10) International Publication Number
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`WO 2007/092473 A2
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`(SI) International Patent Classification:
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`Not classified
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`(2I) International Application Number:
`PCT/US2007/003209
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`(22) International Filing Date: 2 February 2007 (02.02.2007)
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`(25) Filing Language:
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`(26) Publication Language:
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`English
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`English
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`(30) Priority Data:
`60/764,420
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`2 February 2006 (02.02.2006)
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`US
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`(71) Applicant (for all designated States except US): THE
`BOARD OF TRUSTEES OF THE LELAND STAN-
`FORD JUNIOR UNIVERSITY [US/US];
`1705 El
`Camino Real, Palo Alto, CA 94306—1106 (US).
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): QUAKE, Stephen
`[US/US]; 636 Alvarado Row, Stanford, CA 94305 (US).
`FAN, Hei-Mun, Christina [CN/US]; 121 Campus Drive,
`Apt 1314a, Stanford, CA 94305 (US).
`
`(74) Agents: ASTON, David, J. et al.; Peters Vemy, LLP, 425
`Sherman Avenue, Suite 230, Palo Alto, CA 94306 (US).
`
`(8I) Designated States (unless otherwise indicated. for every
`kind of national protection available): AE, AG, AL, AM,
`AT, AU, AZ, BA, BB, BG, BR, BW, BY, BZ, CA, CH, CN,
`CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, EG, ES, FI,
`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, LV, LY, MA, MD, MG, MK, MN, MW, MX, MY,
`MZ, NA, NG, NI, NO, NZ, OM, PG, PH, PL, PT, RO, RS,
`RU, SC, SD, SE, SG, SK, SL, SM, SV, SY, TJ, 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, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI,
`FR, GB, GR, IIU, IE, IS, IT, LT, LU, LV, MC, NL, PL, PT,
`RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA,
`GN, GQ, GW, ML, MR, NE, SN, TD, TG).
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`Published:
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`without international search report and to be republished
`upon receipt of that report
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`For two-letter codes and other abbreviations, refer to the ”Guid-
`ance Notes on Codes and Abbreviations ” appearing at the begin-
`ning of each regular issue of the PCT Gazette.
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`(54) Title: NON—INVASIVE FETAL GENETIC SCREENING BY DIGITAL ANALYSIS
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`ood containing fetal DNA is diluted to a nom-
`(57) Abstract: The present methods are exemplified by a process in which maternal b
`c inal value of approximately 0.5 genome equivalent of DNA per reaction sample. Digital PCR is then be used to detect aneuploidy,
`N such as the trisomy that causes Down Syndrome. Since aneuploidies do not present a mutational change in sequence, and are merely
`21 change in the number of chromosomes, it has not been possible to detect them in a
`~etus without resorting to invasive techniques
`such as amniocentesis or chorionic villi sampling. Digital amplification allows the detection of aneuploidy using massively parallel
`amplification and detection methods, examining, e.g., 10,000 genome equivalents.
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`WO 2007/092473
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`PCT/US2007/003209
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`NON-INVASIVE FETAL GENETIC SCREENING BY DIGITAL ANALYSIS
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`Inventors: Stephen Quake, Hei-Mun Christina Fan
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`CROSS-REFERENCE TO RELATED APPLICATIONS
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`This application claims priority from U.S. Provisional Patent Application No. 60/764,420
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`filed on February 2, 2006, which is hereby incorporated by reference in its entirety.
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`STATEMENT OF GOVERNMENTAL SUPPORT
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`This invention was made with U.S. Government support. The U.S. Government may
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`have certain rights in this invention.
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`REFERENCE TO SEQUENCE LISTING
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`Applicants assert that the paper copy of the Sequence Listing is identical to the Sequence
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`Listing in computer readable form found on the accompanying computer disk. Applicants
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`incorporate the contents of the sequence listing by reference in its entirety.
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`Field of the Invention
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`BACKGROUND OF THE INVENTION
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`The present invention relates to the field of fetal genetic screening and to the field of
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`quantitative nucleic acid analysis.
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`Related Art
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`It is now recognized that fetal DNA sheds from the placenta and mixes with the mother’s
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`blood at fairly high levels — between 3% and 6% of DNA in the mother’s blood is from the fetus.
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`This observation has been used in conjunction with PCR assays for a variety of fetal genetic
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`screens — gender, Rh, and thalassemia. However, the technique remains limited for two primary
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`reasons: first, the PCR assays trade off sensitivity for specificity, making it difficult to identify
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`particular mutations, and second, the most common genetic disorder, DoWn Syndrome, is a
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`chromosomal trisomy and therefore cannot be detected by conventional PCR in a mixed sample.
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`It has now been found that these problems can be solved by quantitative examination of
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`large numbers of chromosome samples through the use of highly scalable techniques. This
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`approach is termed here “digital analysis,” and involves the separation of the extracted genomic
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`material into discrete units so that the detection of a target sequence (e.g., chromosome 21) may
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`be simply quantified as binary (O, 1) or simple multiples, 2, 3, etc. The primary example of a
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`technique that can be used to yield such “digital” results is “digital PCR,” which allows efficient
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`amplification from single molecules, followed by subsequent quantitative analysis. Digital PCR,
`as the term is used here, refers to a quantitative, limited dilution of a nucleic acid sample, such as
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`into multiwell plates, then the amplification of a nucleic acid molecule in a well, which due to
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`the dilution, should be either 0 or 1 molecule. Digital PCR using multiwell plates has been used
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`previously to detect rare mutations by either serial analysis of single molecule (i.e., clonal)
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`amplicons (Vogelstein B, Kinzler KW. Proc Natl Acad Sci USA. 1999 Aug 3; 96 (16): 9236-
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`41) or by enhancing the sensitivity of differential amplification
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`(http://www.fluidigm.com/didIFC.htm). Described below is an invention whereby digital PCR
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`can be applied to noninvasive fetal diagnostics in order to detect fetal mutations with specificity
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`and sensitivity beyond what is possible with conventional PCR analysis.
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`Furthermore, as also described in connection with the invention described below, digital
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`PCR can be used to detect aneuploidy, such as the trisomy that causes Down Syndrome. Since
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`aneuploidies do not present a mutational change in sequence, and are merely a change in the
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`number of chromosomes, it has not been possible to detect them in a fetus without resorting to
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`invasive techniques such as amniocentesis or chorionic villi sampling (Science 309, 2 September
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`2005 pp. 1476-8).
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`Another form of digital PCR has been described as emulsion PCR, which has been used
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`to prepare small beads with clonally amplified DNA — in essence, each bead contains one
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`amplicon of digital PCR. (Dressman et al, Proc Natl Acad Sci U S AJOO, 8817 (Jul 22, 2003)).
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`Another form of Digital PCR can be carried out using microfluidics. In this embodiment,
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`described below, DNA is diluted and separated into small, discrete samples for forming reaction
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`samples by a series of channels and valves.
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`An example of a suitable method for single molecule analysis that may be adapted to the
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`present methods is given in Braslavsky et al., “Sequence information can be obtained from single
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`DNA molecules, Proc. Nat. Acaa’. Sci. 100(7): 39606964 (2003), which uses sequential
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`incorporation of labeled nucleotides onto an immobilized single stranded DNA template and
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`monitoring by fluorescent microscopy.
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`Another aspect of the relevant art involves sample preparation in order to carry out the
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`present processes. That is, the fetal DNA may be enriched relative to maternal DNA. Chan, et
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`31., “Size Distribution of Maternal and Fetal DNA in Maternal Plasma,” Clin. Chem. 50(1): 88—
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`92 (2004) reports that plasma DNA molecules are mainly short DNA fragments. The DNA
`fragments in the plasma of pregnant women are significantly longer than DNA fragments from
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`non-pregnant women, and longer than fetal DNA.
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`Related Publications and Patents
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`Vogelstein et 211., “Digital Amplification,” US 6,440,705, issued Aug. 27, 2002, discloses
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`the identification of pre—defined mutations expected to be present in a minor fraction of a cell
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`population.
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`Lo, “Fetal DNA in Maternal Plasma: Biology and Diagnostic Applications,” Clin. Chem.
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`46: 1903-1906 (2000) discloses the demonstration of fetal DNA in maternal plasma. The authors
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`found a mean fractional level of 3.4% fetal DNA in maternal DNA in plasma during early
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`pregnancy. The authors report detection of the RID gene and microsatellite polymorphisms in
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`the plasma of pregnant women.
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`Li et 9.1., “Detection of Patemally Inherited Fetal Point Mutations for B-Thalassemia
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`Using Size Fractionated Cell-Free DNA in Maternal Plasma,” J. Amer. Med. Assoc. 293:843-849
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`(Feb. 16, 2005) discloses that the analysis of cell-free fetal DNA in maternal plasma has proven
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`to be remarkably reliable for the assessment of fetal loci absent from the maternal genome, such
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`as Y-chromosome specific sequences or the RhD gene in pregnant women who are Rh-negative.
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`The authors report on the extraction and size fractionation of maternal plasma DNA using
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`agarose gel electrophoresis. Then, peptide—nucleic acids (PNA) were used to bind specifically to
`a maternal allele to suppress PCR amplification of the of the wild type maternal allele, thereby
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`enriching for the presence of paternally inherited mutant sequences. Four distinct point
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`mutations in the B-globin gene were examined. It was found that the PNA step was necessary for
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`the detection of mutant alleles using allele specific PCR.
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`Lo et al., “Quantitative Analysis of Fetal DNA in Maternal Plasma and Serum:
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`Implications for Noninvasive Prenatal Diagnosis,” Am. .1 Hum. Genet. 62:768—775 (1998)
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`discloses a real-time quantitative PCR assay to measure the concentration of fetal DNA in
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`maternal plasma and serum. The authors found a mean of 25.4 genome equivalents/ml of fetal
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`DNA in early pregnancy. This corresponds to about 3.4% of total DNA in early pregnancy.
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`Chan et al., “Size Distribution of Maternal and Fetal DNA in Maternal Plasma,” Clin.
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`Chem. 50:89-92 (Jan. 2004) investigated the size distribution of plasma DNA in non-pregnant
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`women and pregnant women, using a' panel of quantitative PCR assays with different amplicon
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`sizes targeting the leptz'n gene. They found that the DNA fragments in the plasma of pregnant
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`women are significantly longer than those in the plasma of non—pregnant women, and the
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`maternal—derived DNA molecules are longer than the fetal-derived ones.
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`Tufan et 111., “Analysis of Cell-Free Fetal DNA from Maternal Plasma and Serum Using a
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`Conventional Multiplex PCR: Factors Influencing Success," Turk. J. Med. Sci. 35: 85—92 (2005)
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`compared the success rates of two different DNA extraction techniques, the heat based direct
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`method and the QIAMP DNA blood mini kit method. The crucial role of PCR optimization was
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`also reported. The authors used the DYSl4 marker for the Y chromosome and the GAPH gene
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`for a control. The QIAMP mini kit was found to give the best results in sex determination
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`analysis using multiplex PCR and ethidium bromide staining on gels.
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`Hromadnikova et 211., “Quantitative analysis of DNA levels in maternal plasma in normal
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`and Down Syndrome pregnancies,” BJWC Pregnancy and Childbirth 2(4): 1-5 (2002),
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`investigated total DNA levels in maternal plasma and found no difference in fetal DNA levels
`between the patients carrying Down Syndrome fetuses and the controls. Real time quantitative
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`PCR analysis was performed using primers to the B-globin gene and the SRY locus.
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`Grundevikk and Rosen, “Molecular Diagnosis of Aneuploidies,” published on line at
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`http://www.molbiotech.chalmers.se/research/ml</mbtk/Molecular%20diagnostics%20ot%20aneu
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`ploidies%20-%20rapport.pdf, suggests that non-invasive methods for detection of aneuploidies
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`(such as Down Syndrome, Edwards Syndrome or extra sex chromosomes) may be carried out on
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`fetal nucleated cells isolated from maternal blood. In their review, the authors also describe
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`quantitative fluorescence polymerase chain reaction (QF-PCR), based on amplification of short
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`tandem repeats Specific for the chromosome to be tested._ They describe tests where DNA was
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`amplified from amniotic or chorionic villus samples. The authors suggest that the STR markers
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`will give PCR products of different size, and these size differences may be studied by analyzing
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`peak sizes in electrophoresis. It is also proposed that quantitative real time PCR may be used to
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`diagnose Down Syndrome by comparing the amount of a gene located on chromosome 12 to the
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`amount of a gene located on another autosomal chromosome. If the ratio of these two genes is
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`1:1, the fetus is normal, but if the ratio of these genes is 3:2, it indicates Down Syndrome. The
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`authors propose the use of Down Syndrome marker DSCR3. They also suggest that the
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`housekeeping gene GAPDH on chromosome 12 can be used as a reference.
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`Poon et al., “Differential DNA Methylation between Fetus and Mother as a Strategy for
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`Detecting Fetal DNA in Maternal Plasma,” Clin. Chem. 48(1): 35-41 discloses the detection of
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`genes or mutations in a fetus where the same mutation or condition is also present in maternal
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`DNA. That is, the use of fetal DNA in maternal plasma is limited due to the low amount of fetal
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`DNA compared to maternal DNA. The authors overcame this limitation by detecting the IGF2-
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`H19 locus, which is maintained in a methylated DNA status in the paternal allele and is
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`unmethylated in the maternal allele. The authors used a bisulfite modification kit whereby
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`unmethylated cytosine residues were converted to uracil. The sequence difference between
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`methylated and unmethylated DNA sequences could be distinguished with different PCR
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`primers. DNA extracted from buffy coat was used.
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`Science 309:1476 (2 Sept. 2005) News Focus “An Earlier Look at Baby’s Genes”
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`describes attempts to develop tests for Down Syndrome using maternal blood. Early attempts to
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`detect Down Syndrome using fetal cells from maternal blood were called “just modestly
`encouraging.” The report also describes work by Dennis L0 to detect the Rh gene in a. fetus
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`where it is absent in the mother. Other mutations passed on from the father have reportedly been
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`detected as well, such as cystic fibrosis, beta—thalassemia, a type of dwarfism and Huntington’s
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`disease. However, these results have not always been reproducible.
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`United States Patent Application 20040137470 to Dhallan, Ravinder S, published July
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`15, 2004, entitled “Methods for detection of genetic disorders,“ describes a method for detecting
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`genetic disorders using PCR of known template DNA and restriction analysis. Also described is
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`an enrichment procedure for fetal DNA. It also describes a method used to detect mutations, and
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`chromosomal abnormalities including but not limited to translocation, transversion, monosomy,
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`trisomy, and other aneuploidies, deletion, addition, amplification, fragment, translocation, and
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`rearrangement. Numerous abnormalities can be detected simultaneously. The method is said to
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`provide a non—invasive method to determine the sequence of fetal DNA from a tissue, such as
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`blood, drawn from a pregnant female, and a method for isolating free nucleic acid from a sample
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`containing nucleic acid.
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`BRIEF SUMMARY OF THE INVENTION
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`The following brief summary is not intended to include all features and aspects of the
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`present invention, nor does it imply that the invention must include all features and aspects
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`discussed in this summary.
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`Briefly, the present invention is directed to a method of differential detection of target
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`sequences in a mixture of maternal and fetal genetic material. One obtains maternal tissue
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`containing both maternal and fetal genetic material. Preferably, the maternal tissue is maternal
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`peripheral blood or blood plasma. The term “plasma” may include plasma or serum. The genetic
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`material may be genomic DNA or RNA, preferably mRNA. In the case of mRNA, one may
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`choose target sequences corresponding to genes that are highly expressed in the placenta for fetal
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`genetic material. The genetic material (e.g., DNA) in each reaction sample is detected with a
`sequence specific reactant directed teat least one oftwo target sequences in the genetic material
`to obtain a detectible reaction product if the target sequence is present in the reaction sample. For
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`example, a probe specific to chromosome 21 is bound to the reaction sample, along with a
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`control probe specific to another chromosome. In most cases, the results will be from maternal
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`DNA, but a small number of results will be obtained from fetal DNA. In order to distinguish
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`random variation from fetal results, a large number of reactions are run, and statistical methods
`are‘applied to the results. The labeling and detection in the present method is used to distinguish
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`the presence or absence of a single target sequence, referred to as “digital analysis,” although it
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`may be performed with sensitive nucleic acid detection methods which distinguish between one
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`and more than one target sequence in a discrete sample. Many fluorescent techniques have this
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`sensitivity. The target sequences are chosen so that a maternal sequence and a fetal sequence are
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`distinguishable, such as two copies of a maternal sequence versus two copies of a fetal sequence.
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`The genetic material thus obtained is distributed into discrete samples, where each
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`sample will contain, on average not more than about one target sequence per sample. The
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`average of one target sequence means that, for practical reasons, the sample will contain,
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`preferably 0.1 to 0.8 genome equivalents per discrete sample, ideally 0.5 genome equivalent per
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`sample. The method may be performed with dilutions whereby more target sequences are
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`detected in samples containing a trisomic or increased copy number of target sequence. That is,
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`if one is analyzing chromosome 21, the mixture may be diluted such that, on average, one may
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`detect two chromosomes present in a maternal DNA, and three chromosomes in a Down
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`Syndrome fetal DNA. Alternatively, the method may be performed with dilutions whereby more
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`reaction samples are positive in this situation. The presence or absence of different target
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`sequences in the discrete samples is detected; and the results are analyzed whereby the number
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`of results from the discrete samples will provide data sufficient to obtain results distinguishing
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`different target sequences. In one aspect, the method involves an analysis of a trisomy. In this
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`method, one of the different target sequences (e.g. chromosome 21) is diploid in maternal
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`genetic material and aneuploid in fetal genetic material and another of the different target
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`sequences (e.g. chromosome 12) is diploid in both maternal and fetal genetic material.
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`The discrete samples are in reaction samples where the target sequences can be analyzed.
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`The reaction samples may be, for example, wells in a microtiter plate, aqueous phases in an
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`emulsion, areas in an array surface, or reaction chambers in a microfluidic device. The reaction
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`samples may be used for PCR analysis of the discrete samples. The discrete samples are
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`contacted with a plurality of PCR primers, including at least one (or one forward and one
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`reverse) primer directed specifically to a maternal control sequence, expected to be the same in
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`both mother and fetus. PCR primers are also directed specifically to a fetal sequence, i.e. one
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`which may be present in both mother and fetus, but is amplified or altered in the fetus. PCR
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`amplification will allow detection of these two different sequences, and, according to the present
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`method, there will be a differential in the case of an abnormal fetal target sequence. The PCR ~
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`method may be (but is not necessarily) quantitative. Quantitative real time PCR, which includes
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`hybridizing target sequences with a nucleic acid having a fluorescent label, may be used. A
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`fluorescent probe hybridizing to the target sequence may also be used. A number of “digital
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`PCR” protocols are known for this purpose, as well as bead—based or emulsion PCR. While
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`florescent probes are readily available and may be used to provide sensitive results, e.g., in
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`FRET combinations, other labeling techniques may be used.
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`The number of discrete samples is chosen according to the results desired. In one aspect,
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`it is preferred that a high degree of statistical significance is obtained, and the number of samples
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`is at least about 10,000. In order to improve statistical confidence, it is preferable to employ
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`large numbers of reactions, preferably between 500 and 100,000, more preferably between
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`10,000 and 100,000 or more reactions, depending on the percentage of fetal DNA present in the
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`mixture. The results to be obtained should be statistically significant for purposes of the analysis
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`conducted, e.g. initial screening, primary diagnosis, etc. A commonly used measure of statistical
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`significance when a highly significant result is desired is p< 0.01, Le, a 99% confidence interval
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`based on a chi-square or t—test.
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`However, as shown below, results can be obtained with less, e.g. on the order of about
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`500 samples, placed in separate reaction samples. Fewer discrete samples may be analyzed
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`where the genetic material is present in a higher concentration in the mixture. The mixture may
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`be enriched for fetal genetic material. One method to enrich plasma DNA for fetal DNA is size
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`separation, whereby a preparation comprising only DNA fragments less than about 300 bp are
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`used for measuring target sequences.
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`A variety of genetic abnormalities may be detected according to the present method,
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`including known alterations in one or more of the genes: CFTR, Factor VIII (F8 gene), beta
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`globin, hemachromatosis, G6PD, neurofibromatosis, GAPDH, beta amyloid, and pyruvate
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`kinase. The sequences and common mutations of these genes are known. Other genetic
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`abnormalities may be detected, such as those involving a sequence which is deleted in a human
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`chromosome, is moved in a translocation or inversion, or is duplicated in a chromosome
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`duplication, wherein said sequence is characterized in a known genetic disorder in the fetal
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`genetic material not present in the maternal genetic material. For example chromosome trisomies
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`may include partial, mosaic, ring, l8, 14, I3, 8, 6, 4 etc. A listing of known abnormalities may
`be found in the OMIM Morbid map, http;//www.ncbi.nimningov/Omim/getmorbid.cgi.
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`In general, the term “aneuploidy” is used to refer to the occurrence of one or more extra
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`or missing chromosomes.
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`In one aspect, the present method of differential detection of target sequences may
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`involve direct sequencing of target sequences the genetic material. Single molecule sequencing,
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`as is known, is further described below. The method may also comprise sequencing of amplified
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`derivatives of the target sequences clones or amplicons of the genetic material. That is, a target
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`sequence in a discrete sample is amplified by PCR, i.e. as an amplicon, or cloned into a vector
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`that is grown up and thereby amplified by obtaining multiple copies of the vector insert.
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`In another aspect,‘the present invention comprises materials selected and combined for
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`carrying out the present methods. Thus is provided a kit for differential detection of target
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`sequences in maternal and fetal DNA in a mixed DNA sample, comprising primers specific for a
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`genetically abnormal sequence and a control sequence, such as two chromosomes, one of which
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`is possibly aneuploid and one of which is presumed diploid; a PCR reaction buffer for forming a
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`PCR reaction sample with the primers in a device having separate reaction samples; and a size
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`separation medium for separating the DNA sample into a fraction having less than about 1000
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`bp. The size separation medium may be gel or centrifugation material for recovering smaller
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`DNA fragments and thus enriching fetal DNA. The kit may further comprise a pair of primers
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`specific to chromosome 21. The kit may filrther comprise the device having separate reaction
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`samples for discrete samples. The device may be a microfluidic device or a microtiter plate
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`having at least 1,000 discrete reaction samples.
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`BRIEF DESCRIPTION OF THE DRAWINGS
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`Figure 1 is a schematic illustration of the present analytical method, showing distribution
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`of genetic material into compartments (1 A), chromosome peaks of different height (1 B), and
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`statistical analysis of chromosomes (IC);
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`Figure 2 is a photograph of a microfluidic chip having 12 panels (numbered 1~12)
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`containing DNA with chromosome 21 labeled;
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`Figure 3 is a photograph of a microfluidic chip having 12 panels (numbered 1~12)
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`containing DNA with chromosome 12 labeled; and
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`Figure 4 is a graph showing results from experiments done using digital analysis of
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`mixed normal and trisomic (Down Syndrome, trisomy 21) DNA.
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`DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
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`Outline
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`I. Overview
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`II. Description of Steps
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`A. Tissue Preparation
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`B. Distribution of DNA molecules
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`C. Detection and Quantification
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`1. Digital PCR Methods
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`2. Bead emulsion PCR
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`3. Microfluidic Dilution with PCR
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`4. Single molecule detection and/or sequencing
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`D. Quantitative evaluation
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`III. Specific applications
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`A. Preparation for tn'somy with frequency analysis.
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`IV. Examples
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`1. Overview
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`The methods and materials described below apply techniques for analyzing numerous
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`nucleic acids contained in a tissue sample (preferably serum or, more preferably, plasma)
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`containing a mixture of DNA from both the mother and the fetus, and allowing detection of
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`small but statistically significant differences.
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`The present invention involves the analysis of maternal blood for a genetic condition,
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`wherein the mixed fetal and maternal DNA in the maternal blood is analyzed to distinguish a
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`fetal mutation or genetic abnormality from the background of the maternal DNA. It has been
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`found that, using a combination of steps, a DNA sample containing DNA from both the mother
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`and the fetus can be analyzed to distinguish a genetic condition present in a minor fraction of the
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`DNA, which represents the fetal DNA. The method employs “digital analysis,” in which the
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`DNA in the sample is isolated to a nominal single target molecule in a small reaction volume.
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`Each sample mixture has a possibility of having distributed in it less than 1 target (i.e., 0 target)
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`or more than one target. Next, the target molecules are detected in each reaction well, preferably
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`as target sequences which are amplified, which may include a quantization of starting copy
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`number of the target sequence, that is, 0, l, 2, 3, etc. A control sequence is used to distinguish an
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`abnormal increase in the target sequence, e.g., a trisonomy. Thus there is a differential detection
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`of target sequences, one of which is chosen to represent a normal genotype present in both
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`mother and offspring, and one of which is chosen for detection of an abnormal genotype in the
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`offspring, where the target sequence in the offspring will be different from that of the mother,
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`e.g. in trisomy.
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`Fig. 1A illustrates an embodiment where quantitative detection, e.g. quantitative real time
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`PCR, is used. Blood 10 is processed to obtain plasma DNA 12, which is diluted and distributed
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`into aliquots 14. These are added to reactions wells 1A through 5D. Shown in the wells are
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`targets representing chromosomes 21 and 22. In well 2A, no target DNA is found; some wells
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`(not shown) may have excess DNA. In well BB, fetal DNA having trisomy 21 (Down Syndrome)
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`is found. The remainder of the wells contains maternal DNA. The DNA is amplified and/or
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`labeled and a quantitative readout is obtained, as shown at 16. Peak 18 representing well 3B will
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`be 50% higher than the peaks from the other well, or the peaks from a reference sequence on
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`chromosome 22. Well A2, lacking either 21 or 22, will have no peak. The peaks are shown at 20.
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`A single run will have numerous random variations, such as wells that have no target sequence,
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`or have duplication through sample variability. Also, samples with no target will clearly result in
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`no peak at all; wells with two or more targets, will give peaks significantly higher than peak 18,
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`i.e., 2X or 2.5 X controls. These results are distinguished by running a multitude of reactions,
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`folIOWed by statistical analysis that can discriminate random variations from true results.
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`Fig. IC illustrates an embodiment where the DNA is distributed in a more dilute fashion
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`(less than 1, or about one half genome equivalents per well). In this case chromosome 21 labels
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`(primers) will generate more positives than chromosome 22 (a diploid chromosome) specific
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`labels (e.g., primers) due simply to the slightly greater abundance of chromosome 21 in a
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`trisomy-containing sample. As shown, some wells will contain positives 20 for both
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`chromosomes, some will contain negatives 22 for both chromosomes, but some will contain
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`blanks 24 for the diploid chromosome and peaks for the trisomic chromosome, due to its greater
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`abundance. The data from a higher peak 18 is not used in this mode. As explained below, this
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`slight difference can be made statistically significant by examining a large number of wells, and
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`by the sensitivity of the present method to a single molecule.
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`Thus, the present method comprises generally the following steps:
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`1. Obtaining a tissue containing DNA from a pregnant subject, which DNA is known to have
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`about 3% fetal DNA. This material is preferably drawn blood, and the circulating DNA is found
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`in the blood plasma, rather than in cells. The blood or plasma may optionally be enriched for
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`fetal DNA by known methods, such as size fractionation to select for DNA fragments less than
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`about 300 bp. Alternatively, maternal DNA, which tends to be larger than about 500 bp may be
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`excluded. Another enrichment step may be to treat the blood sample with formaldehyde, as
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`described in Dhallan et a1. “Methods to Increase the Percentage of Free Fetal DNA Recovered
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`From the Maternal Circulation,” J.Arn. Med. Soc. 291(9): 1114-] 119 (March 2004).
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`2. Distributing single DNA molecules fiom this sample to a number of discrete reaction samples,
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`where the number of reaction samples is selected to give a statistically significant result for the
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`number of copies of a target in the DNA molecules. Further, the reaction sample is confined to a
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`small volume to bring the reaction molecules into close approximation. The amount of DNA
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`molecule per reaction sample is preferably on the order of one copy of the chromosome of
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`interest equivalent per reaction sample.
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`3. Detecting the presence of the target in the DNA in a large number of reaction samples,
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`preferably with a sequence specific technique such as highly multiplexed short read sequencing
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`or a PCR reaction wherein the PCR product is labeled to give a convenient quantitative read out.
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`The detection step is referred to here as “digital PCR” and may be carried out by a variety of
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`methods, such as (a) by PCR on samples diluted into individual wells of a microtiter plate; (b)
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`PCR on samples diluted into emulsions containing primers immobilized to beads; or (0) PCR on
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`samples trapped in a microfluidic chamber; and
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`4. Quantitative analysis of the detection of the maternal and fetal target seguences. In some cases
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`this may include targets to different regions, such as probes to a target on a chromosome
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`suspected of being present in an abnormal copy number (tn'sonomy) compared to a normal
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`diploid