USOO7888017B2
`
`(12) United States Patent
`
`Quake et a1.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 7,888,017 82
`Feb. 15, 2011
`
`(54) NON-INVASIVE FETAL GENETIC
`SCREENING BY DIGITAL ANALYSIS
`
`(75)
`
`Inventors: Stephen Quake. Stanford. CA (US):
`[lei-Mun Christina Fan. Stanford CA
`(US)
`
`(73) Assignee: The Board of Trustees of the Leland
`Stanford Junior University. Palo Alto.
`CA (US)
`
`( " ) Notice:
`
`Subject to any disclaimer. the tenn ol'this
`patent is extended or adjusted under 35
`U.S.(‘. 154(1)) by 65 days.
`
`(21) Appl. No.: 11/701,686
`
`(22)
`
`Filed:
`
`Feb. 2. 2007
`
`(65)
`
`Prior Publication Data
`
`US 2007/0202525 Al
`
`Aug. 30. 2007
`
`Related U.S. Application Data
`
`(60) Provisional application No. 60/764.420. filed on Feb.
`2. 2006.
`
`(51)
`
`Int. Cl.
`(2006.01)
`C12Q [/68
`(2006.01)
`CIZP 19/34
`(52) US. (.‘l.
`......................... 435/6: 435/91.1: 435/912:
`435/9121: 435/915: 435/915]
`(58) Field of Classification Search ....................... None
`See application] iile for complete search history.
`
`(56)
`
`References Cited
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`(Continued)
`
`Primarv ExamineriCaria Myers
`(74) .4Home): Agent. or Firm—David J. Aston: Peters V'emy.
`LLP
`
`(57)
`
`ABSTRACT
`
`The present methods are exemplified by a process in which
`maternal blood containing fetal DNA is diluted to a nominal
`value ol‘approximately 0.5 genome equivalent of DNA per
`reaction sample. Digital 1’(‘R is then be used to detect aneu-
`ploidy. such as the trisomy that causes Down Syndrome.
`Since aneuploidies do not present a mutational change in
`sequence. and are merely a change in the number ofchnomo-
`somes.
`it has not been possible to detect them in a fetus
`without resorting to invasive techniques such as amniocente-
`sis or chorionic vilii sampling. Digital amplification allows
`the detection 01' aneuploidy using massively parallel amplifi-
`cation and detection methods. examining. e.g.. 10.000
`genome equivalents.
`
`28 Claims. 5 Drawing Sheets
`
`Petitioner Sequenom - EX. 1008, p. 1
`
`Petitioner Sequenom - Ex. 1008, p. 1
`
`

`

`US 7,888,017 BZ
`Page 2
`
`OTHER PUBLICATIONS
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`Y.M. Dennis Lo. et al.. “Quantitative Analysis of Fetal NA in Mater-
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`Conicotropin-releasing Hormone mRNA in Maternal Plasma Is
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`727-731.
`ldo Braslavsky. et al.. “Sequence infortnation can be obtained from
`single DNA molecules." PNAS. Apr. 2003. vol. 100. No. 7. 3960-
`3964.
`Jun Zhu. et al.. “Single Molecule Profiling ofAllcmative Pre-mRNA
`Splicing.” Science. Aug. 2003. vol. 301. 836-838.
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`fluorescent magnetic particles for detection and enumeration of
`genetic variations." PNAS. Jul. 2003. vol. 100. No. 15. 8817-8822.
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`long Wook Hong. et al.. “Molecular biology on a microfluidic chip.‘
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`Joshua S. Marcus. et al.. "Parallel Picoliter RT-PCR Assays Using
`Microfluidics.” Analytical Chemistry. Feb. I. 2006. vol. 78. No. 3.
`956-958.
`Y. M. Dennis Lo. et al.. “Plasma placental RNA allelic ratio permits
`noninvasive prenatal chromosomal aneuploidy detection." Nature
`Medicine. Jan. 2007. 1-6.
`Joshua S. Marcus. et al.. “Microfiuidic Single-Cell mRNA Isolation
`and Analysis." American Chemical Society. Mar. 2006. p. A-F.
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`Down syndrome critical region. bridges between fi-amyloid produc-
`tion and tau phosphorylation in Alyheimer disease." Human Molecu-
`lar Genetics. Nov. 29. 2006. vol. 16. No. I. 15-23.
`Y. M. Dennis Lo. et al.. “Prenatal diagnosis: progress through plasma
`nucleic acids.” Nature. Jan. 2007. vol. 8. 71-76.
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`Tetsuya S. Tanaka. et al.. “Genome-wide expression profiling of
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`Fiona M. F. Lun. cta1.. “Microfiuidics Digital PCR Reveals a Higher
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`32.13116-13121.
`Haissam Rahil. et al.. Rapid detection of common autosomal
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`Bernhard Zimmermann. “Molecular Diagnosis in Prenatal Medi-
`cine." PhD. Thesis. 2004. Only Chapter I (Introduction). pp. 1-19.
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`Jay Shcndurc. et al.. "Next-generation DNA sequencing." Nature.
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`11. Christina Fan. et al.. "Noninvasive diagnosis of fetal aneuploidy
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`Plasma Permits Ready Detection of Fetal DNA Polymorphisms."
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`11. Christina Fan. ct al.. “Detection of Ancuploidy with Digital
`Polymerase Chain Reaction." Analytical Chemistry. Oct. 1. 2007.
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`H. Christina Fan. et al.. "Microfiuidic digital PCR enables rapid
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`Leutwyler. K.. Mapping Chromosomes 21. Scientific American. May
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`from a sample of genes." Gene 2005. 357(2):127-143.
`International Search Report. International Application No. PCT/US
`09/‘57136. Mar. 16. 20l0.
`
`‘ cited by examiner
`
`Petitioner Sequenom - EX. 1008, p. 2
`
`Petitioner Sequenom - Ex. 1008, p. 2
`
`

`

`US. Patent
`
`Feb. 15, 2011
`
`Sheet 1 of5
`
`US 7,888,017 82
`
`5
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`
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`
`
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`
`Fig. 1B
`
`Petitioner Sequenom - EX. 1008, p. 3
`
`Petitioner Sequenom - Ex. 1008, p. 3
`
`

`

`US. Patent
`
`Feb. 15, 2011
`
`Sheet 2 of5
`
`US 7,888,017 B2
`
`IAIAWAVAIlVAWAWAVAWAVAVNAWAVA
`
`16 lA-AIAIAIA-lIAIAIAIA_€IAIAIAIAIAIAIAIAIA
`
`
`
`Fig. 1C
`
`Petitioner Sequenom - EX. 1008, p. 4
`
`Petitioner Sequenom - Ex. 1008, p. 4
`
`

`

`US. Patent
`
`Feb. 15, 2011
`
`Sheet 3 of5
`
`US 7.888017 32
`
`
`
`Chr21 FAM
`
`Fig. 2
`
`Petitioner Sequenom - EX. 1008, p. 5
`
`Petitioner Sequenom - Ex. 1008, p. 5
`
`

`

`US. Patent
`
`Feb. 15. 2011
`
`Sheet 4 of5
`
`US 7,888,017 82
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`
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`Chr 12 HEX
`
`Fig. 3
`
`Petitioner Sequenom - EX. 1008, p. 6
`
`Petitioner Sequenom - Ex. 1008, p. 6
`
`

`

`U.S. Patent
`
`Feb. 15. 2011
`
`Sheet 5 of5
`
`US 7,888,017 82
`
`(stdev)
`ConfidenceInterval
`
`
`Percent Down’s DNA
`
`Fig. 4
`
`Petitioner Sequenom - EX. 1008, p. 7
`
`Petitioner Sequenom - Ex. 1008, p. 7
`
`

`

`US 7,888,017 32
`
`l
`NON-[WASIVE FETAL GENETIC
`
`
`SCREENING BY DIGITAL ANALYSIS
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`This application claims priority from US Provisional
`Patent Application No. 60/764.420 filed on Feb. 2. 2006.
`which is hereby incorporated by reference in its entirety.
`FEDERALLY—SPONSORED RESEARCH OR
`DEVEI..OPMF.NT
`
`This invention was made with US. Government support
`under contract 0535870 awarded by the National Science
`Foundation. The US. (iovemment has certain rights in this
`invention
`
`REFERENCE TO SEQUENCE LISTING
`
`Applicants assert that the paper copy of the Sequence List-
`ing is identical to the Sequence Listing in computer readable
`form found on the accompanying computer disk. Applicants
`incorporate the contents of the sequence listing by reference
`in its entirety.
`BACKGROUND OF THE INVENTION
`
`1. Field of the Invention
`The present invention relates to the field of fetal genetic
`screening and to the field ofquantitative nucleic acid analysis.
`2. Related Art
`It is now recognized that fetal DNA sheds from the placenta
`and mixes with the mother‘s blood at fairly high levels—
`between 3% and 6% of DNA in the mother‘s blood is from the
`fetus. ‘lhis observation has been used in conjunction with
`PCR assays for a variety offetal genetic screensigender. Rh.
`and thalassemia. Ilowever. the technique remains limited for
`two primary reasons: first. the PCR assays trade offsensitivity
`for specificity. making it difficult to identify particular muta-
`tions. and second. the most conunon genetic disorder. Down
`Syndrome. is a chromosomal trisomy and therefore cannot be
`detected by conventional PCR in a mixed sample.
`It has now been found that these problems can be solved by
`quantitative examination of large numbers of chromosome
`samples through the use of highly scalable techniques. This
`approach is termed here “digital analysis." and involves the
`separation of the extracted genomic material into discrete
`units so that the detection ofa target sequence (e.g.. chromo-
`some 21) may be simply quantified as binary (0. l )or simple
`multiples. 2. 3. etc. The primary example of a technique that
`can be used to yield such “digital” results is “digital PCR.”
`which allows efficient amplification from single molecules.
`followed by subsequent quantitative analysis. Digital PCR. as
`the term is used here. refers to a quantitative. limited dilution
`ofa nucleic acid sample. such as into multiwell plates. then
`the amplification of a nucleic acid molecule in a well. which
`due to the dilution. should be either 0 or 1 molecule. Digital
`PCR using multiwell plates has been used previously to detect
`rare mutations by either serial analysis of single molecule
`(i.e.. clonal) amplicons (Vogelstein B. Kinzler K W. Proc Natl
`Acad Sci USA. 1999 Aug. 3: 96(16): 9236-41 )or by enhanc-
`ing the sensitivity of differential amplification (world wide
`web address fiuidigm.com/idid/lFC.htm). Described below is
`an invention whereby digital PC R can be applied to noninva-
`sive fetal diagnostics in order to detect fetal mutations with
`specificity and sensitivity beyond what is possible with con-
`ventional PCR analysis.
`
`In
`
`15
`
`2t]
`
`30
`
`35
`
`4(1
`
`45
`
`_
`
`55
`
`60
`
`2
`Furthermore. as also described in connection with the
`invention described below. digital PCR can be used to detect
`aneuploidy. such as the trisomy that causes Down Syndrome.
`Since aneuploidies do not present a mutational change in
`sequence. and are merely a change in the number ofchromo-
`somes.
`it has not been possible to detect them in a fetus
`without resorting to invasive techniques such as anmiocente-
`sis or chorionic villi sampling (Science 309. 2 Sep. 2005 pp.
`1476-8).
`Another form of digital PCR has been described as emul-
`sion PCR. which has been used to prepare small beads with
`clonally amplified DNAriin essence. each bead contains one
`amplicon ofdigital PCR. (Dressman et al. l’roc Natl Acad Sci
`USA. 100. 8817 (Jul. 22. 2003)).
`Another form of Digital PCR can be canied out using
`microfluidics. In this embodiment. described below. DNA is
`diluted and separated into small. discrete samples for forming
`reaction samples by a series of channels and valves.
`An example of a suitable method for single molecule
`analysis that may be adapted to the present methods is given
`in Braslavsky el al.. “Sequence information can be obtained
`from single DNA molecules. Proc. Nat. Acad. Sci. 100(7):
`3960-3964 (2003). which uses sequential incorporation of
`labeled nucleotides onto an immobili7ed single stranded
`DNA template and monitoring by fluorescent microscopy.
`Another aspect of the relevant art involves sample prepa-
`ration in order to carry out the present processes. That is. the
`fetal DNA may be enriched relative to maternal DNA. Chan.
`et al.. “Size Distribution of Maternal and Fetal DNA in Mater-
`nal Plasma." Clin. Chem. 50(1): 88-92 (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 non-pregnant
`women. and longer than fetal DNA.
`Related Publications and Patents
`Vogelstein et al.. “Digital Amplification.“ US Pat No.
`6.440.706. issued Aug. 27. 2002. discloses the identification
`of pre-defined mutations expected to be present in a minor
`fraction of a cell population.
`Lo. “Fetal DNA in Matemal Plasma: Biology and Diag-
`nostic Applications.“ (.‘Iin. (Them. 46:1903—1906 (2000) dis-
`closes the demonstration of fetal DNA in matemal plasma.
`The authors found a mean fractional level of 3.4% fetal DNA
`in maternal DNA in plasma during early pregnancy. The
`authors report detection of the RhD gene and microsatellite
`polymorphisms in the plasma of pregnant women.
`Li et al.. “Detection of Patemally Inherited Fetal Point
`Mutations for B-Thalassemia Using Size Fractionated Cell-
`Free DNA in Maternal Plasma.“ J. Amer: Med. Assoc. 293:
`843-849 (Feb. 16. 2005) discloses that the analysis of cell-
`ti'ee fetal DNA in matemal plasma has proven to be
`remarkably reliable for the assessment of fetal loci absent
`from the maternal genome. such as Y-chromosome specific
`sequences or the RhD gene in pregnant women who are
`Rh-negative. The authors report on the extraction and size
`fractionation ofmatemal plasma DNA using agarose gel elec-
`trophoresis. Then. peptide-nucleic acids (PNA) were used to
`bind specifically to a maternal allele to suppress PCR ampli-
`fication ofthe ofthe wild type matemal allele. thereby enrich-
`ing for the presence ofpaternally inherited mutant sequences.
`Four distinct point mutations in the B-globin gene were exam-
`ined. It was fotmd that the PNA step was necessary for the
`detection of mutant alleles using allele specific PCR.
`Lo et al.. “Quantitative Analysis of Fetal DNA in Maternal
`Plasma and Serum: Implications for Noninvasive Prenatal
`Diagnosis.".4m. J. Hum. Genet. 62:768-775 (1998) discloses
`
`Petitioner Sequenom - EX. 1008, p. 8
`
`Petitioner Sequenom - Ex. 1008, p. 8
`
`

`

`US 7,888,017 32
`
`3
`a real-time quantitative PCR assay to measure the concentra-
`tion of fetal DNA in maternal plasma and serum. The authors
`found a mean of25.4 genome equivalents/ml of fetal DNA in
`early pregnancy. This corresponds to about 3.4% of total
`DNA in wrly pregnancy.
`Chan et al.. “Size Distribution of Maternal and Fetal DNA
`in Maternal Plasma." Clin. Chem. 50:89-92 (January 2004)
`investigated the size distribution of plasma DNA in non-
`pregnant women and pregnant women. using a panel ofquan-
`titative PCR assays with different amplicon sizes targeting
`the leptin gene. They found that the DNA fragments in the
`plasma ofpregnant women are significantly longer than those
`in the plasma of non-pregnant women. and the matemal-
`derived DNA molecules are longer than the fetal-derived
`ones.
`
`Tufan et al.. “Analysis ofCell-Free Fetal DNA from Mater-
`nal Plasma and Serum Using a Conventional Multiplex PCR:
`Factors Influencing Success." 7i/rk. J. Med. Sci. 35: 85—92
`(2005) compared the success rates of two different DNA
`extraction techniques. the heat based direct method and the
`QIAMP DNA blood mini kit method. The crucial role of PCR
`optimization was also reported The authors used the DYSI4
`marker for the Y chromosome and the GAP“ gene for a
`control. The QIAMP mini kit was found to give the best
`results in sex determination analysis using multiplex PCR and
`ethidium bromide staining on gels.
`Hromadnikova et al.. “Quantitative analysis of DNA levels
`in maternal plasma in normal and Down Syndrome pregnan-
`cies.“ BMCPregnanqvand Childbirth 2(4): 1-5 (2002). inves-
`tigated 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 quanti-
`tative PCR analysis was performed using primers to the
`B-globin gene and the SRY locus.
`(irundevikk and Rosen. “Molecular Diagnosis of Aneup-
`loidies." published on line at world wide web address mol-
`biotcch.chalmerssdresearch/mk/mbtk/
`Molecular%20diagnostics%20 ol%20 aneuploidies%20-
`%20rapport.pdf. suggests that non-invasive methods for
`detection ofaneuploidies (such as Down Syndrome. Edwards
`Syndrome or extra sex chromosomes) may be carried out on
`fetal nucleated cells isolated from matemal blood. In their
`review. the authors also describe quantitative fluorescence
`polymerase chain reaction (QF-PCR). based on amplification
`of shon tandem repeats specific for the chromosome to be
`tested. They describe tests where DNA was amplified from
`amniotic or chm-ionic villus samples. The authors suggest that
`the STR markers will give PCR products ofdifferent size. and
`these size differences may be studied by analyzing peak sizes
`in electrophoresis. It is also proposed that quantitative real
`time PCR may be used to diagnose Down Syndrome by
`comparing the amount ofa gene located on chromosome 12 to
`the amount ofa gene located on another autosomal chromo-
`some. Ifthe ratio ofthese two genes is l: l. the fetus is normal.
`but if the ratio of these genes is 3:2. it indicates Down Syn-
`drome. The authors propose the use of Down Syndrome
`marker DSCRS. They also suggest that the housekeeping
`gene (iAPDl-l on chromosome l2 can be used as a reference.
`l’oon et al.. “Di lferent ial DNA Methylation between Fetus
`and Mother as a Strategy for Detecting Fetal DNA in Mater-
`nal Plasma." Clin. Chem. 48(1): 35-41 discloses the detection
`of genes or mutations in a fetus where the same mutation or
`condition is also present in maternal DNA. That is. the use of
`fetal DNA in maternal plasma is limited due to the low
`amount of fetal DNA compared to matemal DNA. The
`authors overcame this limitation by detecting the lGF2-l—l l 9
`locus. which is maintained in a methylated DNA status in the
`
`I“
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`IS
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`20
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`30
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`35
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`4f]
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`45
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`_
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`55
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`60
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`65
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`4
`paternal allele and is unmethylated in the matemal allele. The
`attthors used a bisulfite modification kit whereby unmethy-
`lated cytosine residues were converted to uracil. The
`sequence difference between methylated and unmethylated
`DNA sequences could be distinguished with different PCR
`primers. DNA extracted from buffy coat was used.
`Science 309: 1476 (2 Sep. 2005) News Focus “An Earlier
`Look at Baby’s Genes" describes attempts to develop tests for
`Down Syndrome using maternal blood. Early attempts to
`detect Down Syndrome using fetal cells li'om matcmal blood
`were called “just modestly encouraging." The report also
`describes work by Dennis L0 to detect the Rh gene in a fettrs
`where it is absent in the mother. Other mutations passed on
`from the father have reportedly been detected as well. such as
`cystic fibrosis. beta-thalassemia. a type ofdwarfrsm and Hun-
`tington‘s disease. However. these results have not always
`been reproducible.
`United States Patent Application 20040137470 to Dhallan.
`Ravinder S. published Jul. 15. 2004. entitled “Methods for
`detection ofgenetic disorders.“ describes a method fordetect-
`ing genetic disorders using PCR ofknown template DNA and
`restriction analysis. Also described is an enrichment proce-
`dure for fetal DNA. It also describes a method used to detect
`mutations. and chromosomal abnormalities including but not
`limited to translocation. transversion. monosomy. trisomy.
`and other aneuploidies. deletion. addition. amplification.
`fragment.
`translocation. and rearrangement. Numerous
`abnormalities can be detected simultaneously. The method is
`said to provide a non-invasive method to determine the
`sequence of fetal DNA from a tissue. such as blood. drawn
`from a pregnant female. and a method for isolating free
`nucleic acid from a sample containing nucleic acid.
`
`BRIEF SUMMARY OF THE INVENTION
`
`The following brief summary is not intended to include all
`features and aspects ofthe present invention. nor does it imply
`that the invention must include all features and aspects dis-
`cussed in this sununary.
`Briefly. the present invention is directed to a method of
`differential detection of target sequences in a mixture of
`matemal and fetal genetic material. One obtains maternal
`tissue containing both maternal and fetal genetic material.
`Preferably. the maternal tissue is maternal peripheral blood or
`blood plasma. The term “plasma“ may include plasma or
`serum. The genetic material may be genomic DNA or RNA.
`preferably mRNA. In the case of mRNA. one may choose
`target sequences corresponding to genes that are highly
`expressed in the placenta for fetal genetic material. The
`genetic material (e.g.. DNA) in arch reaction sample is
`detected with a sequence specific reactant directed to at least
`one of two target sequences in the genetic material to obtain
`a detectible reaction product ifthe target sequence is present
`in the reaction sample. For example. a probe specific to chro-
`mosome 21 is bound to the reaction sample. along with a
`control probe specific to anotherchromosome. In most cases.
`the results will be from matemal DNA. but a small number of
`results will be obtained from fetal DNA. In order to distin-
`guish 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 the presence or absence of a single target
`sequence. referred to as “digital analysis." although it may be
`performed with sensitive nucleic acid detection methods
`which distinguish between one and more than one target
`sequence in a discrete sample. Many fluorescent techniques
`have this sensitivity. The target sequences are chosen so that
`
`Petitioner Sequenom - EX. 1008, p. 9
`
`Petitioner Sequenom - Ex. 1008, p. 9
`
`

`

`US 7,888,017 32
`
`In
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`15
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`2t]
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`5
`a malcmal sequence and a fetal sequence are distinguishable.
`such as two copies of a matemal sequence versus two copies
`of a fetal sequence.
`The genetic material thus obtained is distributed into dis-
`crete samples. where each sample will contain. on average not
`more than about one target sequence per sample. The average
`of one target sequence means that. for practical reasons. the
`sample will contain. preferably 0.1 to 0.8 genome equivalents
`per discrete sample.
`ideally 0.5 genome equivalent per
`sample. The method may be performed with dilutions
`whereby more target sequences are detected in samples con-
`taining a trisomic or increased copy number of target
`sequence. That is. if one is analyzing chromosome 2|. the
`mixture may be diluted such that. on average. one may detect
`two chromosomes present in a malcmal DNA. and three
`chromosomes in a Down Syndrome fetal DNA. Altematively.
`the method may be performed with dilutions whereby more
`reaction samples are positive in this situation. The presence or
`absence of different target sequences in the discrete samples
`is detected: and the results are analyzed whereby the number
`of results from the discrete samples will provide data suffi-
`cient
`to obtain results distinguishing different
`target
`sequences. In one aspect. the method involves an analysis of
`a trisomy. In this method, one ofthedifferent target sequences
`(e.g. chromosome 21) is diploid in matemal genetic material
`and anettploid in fetal genetic material and another of the
`different target sequences (e.g. chromosome 12) is diploid in
`both maternal and fetal genetic material.
`The discrete samples are in reaction samples where the
`target sequences can be analyzed. 'lhe reaction samples may
`be. for example. wells in a microtiter plate, aqueous phases in
`an emulsion. areas in an array surface. or reaction chambers in
`a microfiuidic device. The reaction samples may be used for
`PCR analysis of the discrete samples. The discrete samples
`are contacted with a plurality of PCR primers. including at
`least one (or one forward and one reverse) primer directed
`specifically to a malcmal control sequence. expected to be the
`same in both mother and fetus. PCR printers are also directed
`specifically to a fetal sequence. i.e. one which may be present
`in both mother and fetus, but is amplified or altered in the
`fetus. PCR amplification will allow detection of these two
`different sequences. and. according to the present method.
`there will be a differential in the case of an abnormal fetal
`target sequence. The PCR method may be (but is not neces-
`sarily) quantitative. Quantitative real
`time PCR. which
`includes hybridizing target sequences with a nucleic acid
`having a fluorescent label. may be used. A fluorescent probe
`hybridizing to the target sequence may also be used. A num-
`ber of“digital PCR” protocols are known for this purpose. as
`well as bead-based oremulsion PCR. While llorcscent probes _
`are readily available and may be used to provide sensitive
`results. e.g..
`in FRET combinations. other labeling tech-
`niques may be used.
`The number ofdiscrete samples is chosen according to the
`results desired. 1n one aspect. it is preferred that a high degree
`of statistical significance is obtained. and the number of
`samples is at least about 10,000. In order to improve statistical
`confidence. it is preferable to employ large numbers of reac-
`tions. preferably between 500 and 100.000. more preferably
`between 10.000 and 100.000 or more reactions. depending on
`the percentage of fetal DNA present in the mixture. The
`results to be obtained should be statistically significant for
`purposes of the analysis conducted. e.g.
`initial screening.
`primary diagnosis. etc. A commonly used measure of statis-
`tical significance when a highly significant result is desired is
`p<0.01. i.e.. a 99% confidence interval based on a chi-square
`or t-test.
`
`6
`However. as shown below. results can be obtained with
`less. e.g. on the orderofabout 500 samples. placed in separate
`reaction samples. Fewer discrete samples may be analyzed
`where the genetic material is present in a higher concentration
`in the mixture. The mixture may be enriched for fetal genetic
`material. One method to enrich plasma DNA for fetal DNA is
`size separation. whereby a preparation comprising only DNA
`fragments less than about 300 bp are used for measuring
`target sequences.
`A variety ofgenetic abnormalities may be detected accord-
`ing to the present method. including known alterations in one
`or more of the genes: CFfR. Factor VIII (F8 gene). beta
`globin.
`hemachromatosis. GéPD.
`neurofibromatosis.
`GAPDH. beta amyloid. and pyruvate kinase. The sequences
`and common mutations of these genes are known. Other
`genetic abnormalities may be detected. such as those involv-
`ing a sequence which is deleted in a human chromosome. is
`moved in a translocation or inversion. or is duplicated in a
`chromosome duplication. wherein said sequence is charac-
`teri 7ed in