llIIlIlllIlIlIllIlIllIllIllIIlllIIllllIIllIIllIllIllIIllIllIIlIlllIllIIlIllIIIIIIIIIIIIIII
`US 20070202525AI
`
`&19) United States
`(12) Patent Application Publication
`Quake et al.
`
`(Io) Pub. No. : US 2007/0202525 A1
`Aug. 30, 2007
`(43) Pub. Date:
`
`(54) NON-INVASIVE FETAL GENETIC
`SCREENING BY DIGITAL ANALYSIS
`
`(75)
`
`Inventors: Stephen Quake, Stanford, CA (US);
`Hei-Mun Christina Fan, Stanford, CA
`(US)
`
`Publication ClassiTication
`
`(51) Int. Cl.
`C12Q 1/68
`C12P 19/34
`(52) U. S. Cl.
`
`(2006. 01)
`(2006. 01)
`
`435/6; 435/91. 2
`
`Correspondence Address:
`PETERS VERNY, L. L. P.
`425 SHERMAN AVENUE
`SUITE 230
`PALO ALTO, CA 94306 (US)
`
`(73) Assignee: The Board of Trustees of the Leland
`Stanford Junior University
`
`(21) Appl. No. :
`
`11/701, 6S6
`
`(22) Filed:
`
`Feb. 2, 2007
`
`Related U. S. Application Data
`
`(60) Provisional application No. 60/764, 420, filed on Feb.
`2, 2006.
`
`(57)
`
`ABSTRACT
`
`The present methods are exemplified by a process in which
`maternal blood containing
`fetal DNA is diluted to a nominal
`value of approximately 0. 5 genome equivalent of DNA per
`reaction sample. Digital PCR is then be used
`to detect
`that causes Down Syn-
`such as the trisomy
`aneuploidy,
`drome. Since aneuploidies
`do not present a mutational
`change in sequence, and are merely a change in the number
`of chromosomes,
`it has not been possible to detect them in
`to invasive
`a fetus without
`such as
`resorting
`techniques
`amniocentesis or chorionic villi sampling. Digital amplifi-
`the detection of aneuploidy using massively
`cation allows
`and detection methods,
`parallel amplification
`examining,
`e. g. , 10, 000 genome equivalents.
`
`10
`
`12
`
`0»
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`21, 21
`22, 22
`
`21, 21
`22, 22
`
`21, 21
`22, 22
`
`21, 21
`22, 22
`
`21, 21
`22, 22
`
`21, 21
`22, 22
`
`21, 21, 21
`22, 22
`
`21, 21
`22, 22
`
`21, 21
`22, 22
`
`21, 21
`22, 22
`
`21, 21
`22, 22
`
`21, 21
`22, 22
`
`21, 21
`22, 22
`
`21, 21
`22, 22
`
`21, 21
`22, 22
`
`21, 21
`22, 22
`
`21, 21
`22, 22
`
`21, 21
`22, 22
`
`21, 21
`22, 22
`
`D
`
`Ariosa Exhibit 1006, p. 1
`
`

`

`Patent Application Publication Aug. 30, 2007 Sheet 1 of 5
`
`US 2007/0202525 A1
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`10
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`12
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`0»
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`0»
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`21, 21
`22, 22
`
`21, 21
`22, 22
`
`21, 21
`22, 22
`
`21, 21
`22, 22
`
`B
`
`21, 21
`22, 22
`
`21, 21
`22, 22
`
`21, 21, 21
`22, 22
`
`21, 21
`22, 22
`
`21, 21
`22, 22
`
`21, 21
`22, 22
`
`21, 21
`22, 22
`
`21, 21
`22, 22
`
`21, 21
`22, 22
`
`21, 21
`22, 22
`
`21, 21
`22, 22
`
`21, 21
`22, 22
`
`21, 21
`22, 22
`
`21, 21
`22, 22
`
`21, 21
`22, 22
`
`D
`
`Fig. 1A
`
`16
`
`20
`
`Chromosome 21
`
`Chromosome 22 (All peaks
`the same height)
`
`Ariosa Exhibit 1006, p. 2
`
`

`

`Patent Application Publication Aug. 30, 2007 Sheet 2 of 5
`
`US 2007/0202525 A1
`
`16
`
`20
`
`Chromosome 21
`
`Chromosome 22 (fewer peaks)
`
`22
`
`24
`
`Ariosa Exhibit 1006, p. 3
`
`

`

`Patent Application Publication Aug. 30, 2007 Sheet 3 of 5
`
`US 2007/0202525 A1
`
`10
`
`12
`
`Chr21 FAM
`
`Ariosa Exhibit 1006, p. 4
`
`

`

`Patent Application Publication Aug. 30, 2007 Sheet 4 of 5
`
`US 2007/0202525 A1
`
`10
`
`12
`
`Ghr 12 HEX
`
`Ariosa Exhibit 1006, p. 5
`
`

`

`Patent Application Publication Aug. 30, 2007 Sheet 5 of 5
`
`US 2007/0202525 A1
`
`o 5
`
`4
`
`tO
`
`Ol c 3
`I O
`C I 2
`
`'Q
`
`1
`
`O
`
`0. 3
`
`0. 4
`
`0. 5
`
`0. 6
`
`Percent Down's DNA
`
`Ariosa Exhibit 1006, p. 6
`
`

`

`US 2007/0202525 A1
`
`Aug. 30, 2007
`
`NON-INVASIVE FETAL GENETIC SCREENING BY
`DIGITAL ANALYSIS
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`from U. S. Provi-
`[0001] This application claims priority
`sional Patent Application No. 60/764, 420 filed on Feb. 2,
`2006, which
`by reference
`incorporated
`is hereby
`in its
`entirety.
`
`STATEMENT OF GOVERNMENTAL SUPPORT
`[0002] This invention was made with U. S. Government
`support. The U. S. Government may have certain rights
`in
`this invention.
`
`REFERENCE TO SEQUENCE LISTING
`the paper copy of the
`[0003] Applicants
`assert
`that
`to the Sequence Listing
`in
`Sequence Listing
`is identical
`com-
`computer readable form found on the accompanying
`the contents of the
`puter disk. Applicants
`incorporate
`listing by reference
`in its entirety.
`sequence
`
`BACKGROUND OF THE INVENTION
`[0004] 1. Field of the Invention
`relates to the field of fetal
`[0005] The present
`invention
`genetic screening and to the field of quantitative nucleic acid
`analysis.
`[0006] 2. Related Art
`[0007]
`It is now recognized that fetal DNA sheds from the
`placenta and mixes with the mother's blood at fairly high
`between 3% and 6% of DNA in the mother's blood
`levels
`is from the fetus. This observation has been used in con-
`junction with PCR assays for a variety of fetal genetic
`the tech-
`screens
`gender, Rh, and thalassemia. However,
`for two primary
`reasons:
`first, the
`limited
`nique remains
`trade ofl' sensitivity
`PCR assays
`for specificity, making
`it
`and second,
`to identify particular mutations,
`the
`difficult
`is a chro-
`most common genetic disorder, Down Syndrome,
`trisomy and therefore cannot be detected by con-
`mosomal
`ventional PCR in a mixed sample.
`[0008]
`It has now been found that these problems can be
`examination of large numbers of
`solved by quantitative
`the use of highly
`chromosome
`scalable
`samples
`through
`is termed here "digital analysis, "
`techniques. This approach
`the separation of the extracted genomic mate-
`and involves
`the detection of a target
`into discrete units
`rial
`so that
`sequence (e. g. , chromosome 21) may be simply quantified
`as binary (0, I) or simple multiples, 2, 3, etc. The primary
`example of a technique
`that can be used
`to yield such
`is "digital PCR, " which allows efficient
`"digital" results
`followed by subse-
`from single molecules,
`amplification
`quent quantitative analysis. Digital PCR, as the term is used
`limited dilution of a nucleic
`here, refers to a quantitative,
`acid sample, such as into multiwell plates, then the ampli-
`fication of a nucleic acid molecule
`in a well, which due to
`the dilution, should be either 0 or I molecule. Digital PCR
`to detect
`using multiwell plates has been used previously
`rare mutations by either serial analysis of single molecule
`(Vogelstein B, Kinzler K W. Proc
`(ke. , clonal) amplicons
`Natl Acad Sci USA. 1999 Aug. 3; 96 (16): 9236-41) or by
`the sensitivity of difl'erential amplification
`enhancing
`(http: //
`
`www. fluidigm. corn/didIFC. htm). Described below
`is an
`invention whereby digital PCR can be applied to noninva-
`in order to detect fetal mutations with
`sive fetal diagnostics
`is possible with
`specificity and sensitivity beyond what
`conventional PCR analysis.
`[0009] Furthermore,
`in connection with
`as also described
`the invention described below, digital PCR can be used to
`detect aneuploidy,
`that causes Down
`such as the trisomy
`Syndrome. Since aneuploidies do not present a mutational
`change in sequence, and are merely a change in the number
`of chromosomes,
`it has not been possible to detect them in
`to invasive
`a fetus without
`such as
`resorting
`techniques
`(Science 309, 2
`amniocentesis or chorionic villi sampling
`Sep. 2005 pp. 1476-8).
`[0010] Another form of digital PCR has been described as
`emulsion PCR, which has been used to prepare small beads
`in essence, each bead con-
`with clonally amplified DNA
`tains one amplicon of digital PCR. (Dressman et al, Proc
`Natl Acad Sci USA. 100, 8817 (Jul. 22, 2003)).
`form of Digital PCR can be carried out
`[0011] Another
`described below,
`using microfluidics.
`In this embodiment,
`DNA is diluted and separated
`into small, discrete samples
`reaction samples by a series of channels and
`for forming
`valves.
`[0012] An example of a suitable method for single mol-
`that may be adapted to the present methods
`ecule analysis
`is
`in Braslavsky et al. , "Sequence information
`can be
`given
`obtained from single DNA molecules, Proc. Nat. Head. Sci.
`100(7): 3960-3964 (2003), which uses sequential
`incorpo-
`ration of labeled nucleotides onto an immobilized
`single
`stranded DNA
`by fluorescent
`and monitoring
`template
`microscopy.
`[0013] Another aspect of the relevant art involves sample
`in order to carry out the present processes. That
`preparation
`is, the fetal DNA may be enriched relative to maternal DNA.
`Chan, et al. , "Size Distribution of Maternal and Fetal DNA
`in Maternal Plasma, "Clin. Chem. 50(1): 88-92 (2004)
`reports that plasma DNA molecules are mainly short DNA
`in the plasma of pregnant
`fragments. The DNA fragments
`longer than DNA fragments
`from
`women are significantly
`non-pregnant women, and longer than fetal DNA.
`
`Related Publications and Patents
`
`[0014] Vogelstein et al. , "Digital Amplification, " U. S. Pat.
`No. 6, 440, 705, issued Aug. 27, 2002, discloses the identi-
`fication of pre-defined mutations expected to be present in a
`minor fraction of a cell population.
`[0015] Lo, "Fetal DNA in Maternal Plasma: Biology and
`Diagnostic Applications, "Clin. Chem. 46:1903-1906 (2000)
`of fetal DNA
`discloses
`in maternal
`the demonstration
`level of 3. 4%
`plasma. The authors found a mean fractional
`fetal DNA in maternal DNA in plasma during early preg-
`report detection of the RhD gene and
`nancy. The authors
`in the plasma of pregnant
`microsatellite
`polymorphisms
`women.
`[0016] Li et al. , "Detection of Paternally
`Inherited Fetal
`for JJ-Thalassemia Using Size Fractionated
`Point Mutations
`Cell-Free DNA in Maternal Plasma, "J. Amer. Med. Assoc.
`that the analysis of
`293:843-849 (Feb. 16, 2005) discloses
`cell-free fetal DNA in maternal plasma has proven
`to be
`reliable for the assessment of fetal loci absent
`remarkably
`
`Ariosa Exhibit 1006, p. 7
`
`

`

`US 2007/0202525 A1
`
`Aug. 30, 2007
`
`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 of maternal plasma DNA using 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
`for the presence of paternally
`enriching
`inherited mutant
`sequences. Four distinct point mutations
`in the JJ-globin
`It was found that the PNA step was
`gene were examined.
`for the detection of mutant alleles using allele
`necessary
`specific PCR.
`[0017] Lo et al. , "Quantitative Analysis of Fetal DNA in
`for Noninvasive
`Maternal Plasma and Serum: Implications
`Prenatal Diagnosis, "Am. J. Hum. Genet. 62:768-775 (1998)
`discloses a real-time quantitative PCR assay to measure
`the
`concentration of fetal DNA in maternal plasma and serum.
`The authors found a mean of 25. 4 genome equivalents/ml of
`to about
`fetal DNA in early pregnancy. This corresponds
`3. 4% of total DNA in early pregnancy.
`[0018] Chan et al. , "Size Distribution of Maternal
`and
`in Maternal Plasma, "Clin. Chem. 50:89-92
`Fetal DNA
`the size distribution of plasma
`(January 2004) investigated
`DNA in non-pregnant women and pregnant women, using a
`panel of quantitative PCR assays with diflerent amplicon
`that the DNA
`the leptin gene. They found
`sizes targeting
`in the plasma of pregnant women are significantly
`fragments
`longer than those in the plasma of non-pregnant women, and
`the maternal-derived DNA molecules are longer
`the
`than
`fetal-derived ones.
`[0019] Tufan et al. , "Analysis of Cell-Free Fetal DNA
`from Maternal Plasma and Serum Using a Conventional
`Multiplex PCR: Factors Influencing Success, "Turk J. Med.
`the success rates of two
`Sci. 35: 85-92 (2005) compared
`the heat based direct
`difl'erent DNA extraction
`techniques,
`method and the QIAMP DNA blood mini kit method. The
`crucial role of PCR optimization was also reported. The
`authors used the DYS14 marker for the Y chromosome and
`the GAPH gene for a control. The QIAMP mini kit was
`in sex determination
`found to give the best results
`analysis
`using multiplex PCR and ethidium bromide staining on gels.
`analysis of
`[0020] Hromadnikova
`et al. , "Quantitative
`DNA levels in maternal plasma
`in normal and Down Syn-
`drome pregnancies, "BMC Pregnancy and Childbirth 2(4):
`1-5 (2002), investigated
`total DNA
`levels
`in maternal
`plasma and found no difference in fetal DNA levels between
`fetuses and the con-
`the patients carrying Down Syndrome
`trols. Real time quantitative PCR analysis was performed
`to the JJ-globin gene and the SRY locus.
`using primers
`and Rosen, "Molecular Diagnosis of
`[0021] Grundevikk
`Aneuploidies, " published on line at http: //www. molbiotech-
`
`. chalmers. se/research/mk/mbtk/
`ploidies%20-
`Molecular%20diagnostics%20of%20aneu
`for
`that non-invasive methods
`%20rapport. pdf, suggests
`detection of aneuploidies
`as Down Syndrome,
`(such
`Edwards Syndrome or extra sex chromosomes) may be
`carried out on fetal nucleated cells isolated from maternal
`blood. In their review, the authors also describe quantitative
`fluorescence polymerase chain reaction (QF-PCR), based on
`amplification of short tandem repeats specific for the chro-
`to be tested. They describe tests where DNA was
`mosome
`from amniotic or chorionic villus samples. The
`amplified
`
`that the STR markers will give PCR prod-
`authors suggest
`ucts of difl'erent
`size, and these size difl'erences may be
`It is also
`studied by analyzing peak sizes in electrophoresis.
`real time PCR may be used to
`that quantitative
`proposed
`the amount of a
`diagnose Down Syndrome by comparing
`gene located on chromosome 12 to the amount of a gene
`located on another autosomal chromosome. If the ratio of
`these two genes is I: I, the fetus is normal, but if the ratio of
`these genes is 3:2, it indicates Down Syndrome. The authors
`propose the use of Down Syndrome marker DSCR3. They
`gene GAPDH on chro-
`also suggest
`that the housekeeping
`mosome 12 can be used as a reference.
`
`[0022] Poon
`"Difl'erential DNA Methylation
`et al. ,
`between Fetus and Mother as a Strategy for Detecting Fetal
`DNA in Maternal Plasma, "Clin. Chem. 48(1): 35-41 dis-
`closes the detection of genes or mutations
`in a fetus where
`the same mutation or condition
`in maternal
`is also present
`DNA. That is, the use of fetal DNA in maternal plasma
`is
`limited due to the low amount of fetal DNA compared
`to
`maternal DNA. The authors overcame
`limitation by
`this
`the IGF2-H19 locus, which
`in a
`detecting
`is maintained
`in the paternal allele and is unm-
`methylated DNA status
`in the maternal allele. The authors used a bisulfite
`ethylated
`modification kit whereby unmethylated
`residues
`cytosine
`to uracil. The sequence difl'erence between
`were converted
`could be
`and unmethylated DNA sequences
`methylated
`distinguished with diflerent PCR primers. DNA extracted
`from buffy coat was used.
`[0023] Science 309:1476 (2 Sep. 2005) News Focus "An
`Earlier Look at Baby's Genes" describes attempts to develop
`using maternal blood. Early
`for Down Syndrome
`tests
`to detect Down Syndrome using fetal cells from
`attempts
`maternal blood were called "just modestly encouraging. "
`The report also describes work by Dennis Lo to detect the
`Rh gene in a fetus where
`in the mother. Other
`it is absent
`mutations passed on from the father have reportedly been
`detected as well, such as cystic fibrosis, beta-thalassemia,
`a
`type of dwarfism and Huntington's
`disease. However,
`these
`results have not always been reproducible.
`[0024] United States Patent Application 20040137470 to
`Jul. 15, 2004, entitled
`Dhallan, Ravinder S, published
`"Methods for detection of genetic disorders, " describes a
`method for detecting genetic disorders using PCR of known
`template DNA and restriction analysis. Also described is an
`for fetal DNA. It also describes a
`enrichment procedure
`abnor-
`method used to detect mutations,
`and chromosomal
`including but not limited to translocation,
`transver-
`malities
`trisomy, and other aneuploidies, deletion,
`sion, monosomy,
`and rear-
`translocation,
`addition, amplification,
`fragment,
`can be detected simul-
`rangement. Numerous abnormalities
`is said to provide a non-invasive
`taneously. The method
`the sequence of fetal DNA from a
`to determine
`method
`tissue, such as blood, drawn from a pregnant
`female, and a
`free nucleic acid from a sample con-
`method for isolating
`taining nucleic acid.
`BRIEF SUMMARY OF THE INVENTION
`[0025] The following brief summary
`to
`is not intended
`include all features and aspects of the present invention, nor
`does it imply that the invention must include all features and
`aspects discussed
`in this summary.
`[0026] Briefly,
`is directed
`the present
`invention
`method of diflerential detection of target sequences
`
`to a
`in a
`
`Ariosa Exhibit 1006, p. 8
`
`

`

`US 2007/0202525 A1
`
`Aug. 30, 2007
`
`3
`
`mixture of maternal and fetal genetic material. One obtains
`tissue containing both maternal and fetal genetic
`maternal
`tissue is maternal periph-
`material. Preferably,
`the maternal
`eral 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
`in the placenta for fetal genetic material.
`highly expressed
`The genetic material (e. g. , DNA) in each 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 if the target sequence is present
`in the reaction sample. For example, a probe specific to
`chromosome 21 is bound to the reaction sample, along with
`a control probe specific to another chromosome.
`In most
`cases, the results will be from maternal DNA, but a small
`number of results will be obtained from fetal DNA. In order
`to distinguish
`from fetal results, a large
`random variation
`number of reactions are run, and statistical methods are
`to the results. The labeling and detection
`in the
`applied
`the presence or
`to distinguish
`is used
`present method
`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 a maternal sequence and a fetal
`such as two copies of a mater-
`sequence are distinguishable,
`two copies of a fetal sequence.
`nal sequence versus
`[0027] The genetic material
`is distributed
`thus obtained
`into discrete 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
`the sample will contain, preferably 0. 1 to 0. 8
`reasons,
`genome equivalents per discrete sample, ideally 0. 5 genome
`equivalent per sample. The method may be performed with
`target sequences are detected
`dilutions whereby more
`in
`samples containing a trisomic or increased copy number of
`target sequence. That is, if one is analyzing chromosome 21,
`the mixture may be diluted such that, on average, one may
`detect two chromosomes present
`in a maternal DNA, and
`in a Down Syndrome fetal DNA. Alter-
`three chromosomes
`the method may be performed with dilutions
`natively,
`whereby more reaction samples are positive in this situation.
`The presence or absence of diflerent
`in the
`target sequences
`is detected; and the results are analyzed
`discrete samples
`the number of results
`from the discrete samples
`whereby
`to obtain results distinguishing
`will provide data sufficient
`In one aspect,
`difl'erent
`sequences.
`the method
`target
`involves an analysis of a trisomy. In this method, one of the
`target sequences (e. g. chromosome 21) is diploid in
`difl'erent
`in fetal genetic
`and aneuploid
`maternal genetic material
`material and another of the diflerent
`target sequences (e. g.
`chromosome 12) is diploid in both maternal and fetal genetic
`material.
`[0028] The discrete samples are in reaction samples where
`the target sequences can be analyzed. The reaction samples
`may be, for example, wells
`in a microtiter plate, aqueous
`phases in an emulsion, areas in an array surface, or reaction
`in a microfluidic device. The reaction samples
`chambers
`may be used for PCR analysis of the discrete samples. The
`samples are contacted with a plurality of PCR
`discrete
`at least one (or one forward and one
`including
`primers,
`reverse) primer directed specifically
`to a maternal control
`
`sequence, expected to be the same in both mother and fetus.
`PCR primers
`are also directed
`to a fetal
`specifically
`sequence, i. e. one which may be present in both mother and
`fetus, but is amplified or altered in the fetus. PCR amplifi-
`cation will allow detection of these two difl'erent sequences,
`there will be a
`and, according
`to the present method,
`in the case of an abnormal
`fetal target sequence.
`difl'erential
`The PCR method may be (but is not necessarily) quantita-
`real time PCR, which includes hybridizing
`tive. Quantitative
`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 number of "digital
`PCR" protocols are known
`for this purpose, as well as
`bead-based or emulsion PCR. While florescent probes are
`to provide
`and may be used
`readily available
`sensitive
`results, e. g. , in FRET combinations,
`tech-
`other
`labeling
`niques may be used.
`[0029] The number of discrete samples
`is chosen accord-
`ing to the results desired. In 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
`to employ
`statistical confidence,
`it is preferable
`improve
`large numbers of reactions, preferably between 500 and
`100, 000, more preferably between 10, 000 and 100, 000 or
`more reactions, depending on the percentage of fetal DNA
`in the mixture. The results to be obtained should be
`present
`for purposes of the analysis con-
`statistically
`significant
`ducted, e. g. initial
`etc. A
`diagnosis,
`screening, primary
`commonly used measure of statistical significance when a
`is p&0. 01, i. e. , a 99%
`is desired
`result
`significant
`highly
`interval based on a chi-square or t-test.
`confidence
`[0030] However, as shown below, results can be obtained
`with less, e. g. on the order of about 500 samples, placed in
`separate reaction samples. Fewer discrete samples may be
`is present
`in a higher
`the genetic material
`analyzed where
`in the mixture. The mixture may be enriched
`concentration
`for fetal genetic material. One method
`to enrich plasma
`DNA for fetal DNA is size separation, whereby a preparation
`less than about 300 bp are
`comprising only DNA fragments
`used for measuring
`target sequences.
`[0031] A variety of genetic abnormalities may be detected
`including known alter-
`to the present method,
`according
`ations in one or more of the genes: CFTR, Factor VIII (F8
`gene), beta globin, hemachromatosis, G6PD, neurofibroma-
`and pyruvate kinase. The
`tosis, GAPDH, beta amyloid,
`sequences and common mutations of these genes are known.
`Other genetic abnormalities may be detected, such as those
`involving a sequence which is deleted in a human chromo-
`some, is moved in a translocation or inversion, or is dupli-
`cated in a chromosome duplication, wherein said sequence
`is characterized
`in a known genetic disorder
`in the fetal
`genetic material not present in the maternal genetic material.
`For example chromosome
`trisomies may
`include partial,
`mosaic, ring, 18, 14, 13, 8, 6, 4 etc. A listing of known
`abnormalities may be found
`in the OMIM Morbid map,
`http://www. ncbi. nlm. nih. gov/Omim/getmorb
`id. cgi.
`[0032]
`In general, the term "aneuploidy"
`is used to refer to
`the occurrence of one or more extra or missing chromo-
`somes.
`[0033] In one aspect, the present method of difl'erential
`detection of target sequences may involve direct sequencing
`of target sequences
`the genetic material. Single molecule
`
`Ariosa Exhibit 1006, p. 9
`
`

`

`US 2007/0202525 A1
`
`Aug. 30, 2007
`
`is further described below. The
`as is known,
`sequencing,
`method may also comprise sequencing of amplified deriva-
`tives of the target sequences clones or amplicons of the
`in a discrete
`genetic material. That is, a target sequence
`is amplified by PCR, i. e. as an amplicon, or cloned
`sample
`into a vector that
`is grown up and thereby amplified by
`obtaining multiple copies of the vector insert.
`[0034]
`In another aspect, the present
`invention comprises
`materials selected and combined for carrying out the present
`methods. Thus is provided a kit for differential detection of
`in maternal and fetal DNA in a mixed DNA
`target sequences
`sample, comprising primers specific for a genetically abnor-
`mal sequence and a control sequence, such as two chromo-
`somes, one of which is possibly aneuploid and one of which
`is presumed diploid; a PCR reaction bufl'er for forming a
`PCR reaction sample with the primers
`in a device having
`separate reaction samples; and a size separation medium for
`into a fraction having
`the DNA sample
`less than
`separating
`about 1000 bp. The size separation medium may be gel or
`for recovering
`smaller DNA frag-
`centrifugation material
`fetal DNA. The kit may further
`ments and thus enriching
`comprise a pair of primers specific to chromosome 21. The
`kit may further comprise the device having separate reaction
`samples for discrete samples. The device may be a microf-
`luidic device or a microtiter plate having at least 1, 000
`discrete reaction samples.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`illustration of the present
`[0035] FIG. 1 is a schematic
`analytical method, showing distribution of genetic material
`(IA), chromosome peaks of diflerent
`into compartments
`height (IB), and statistical analysis of chromosomes
`(IC);
`[0036] FIG. 2 is a photograph of a microfluidic
`chip
`(numbered 1-12) containing DNA with
`having 12 panels
`chromosome 21 labeled;
`[0037] FIG. 3 is a photograph of a microfluidic
`chip
`(numbered 1-12) containing DNA with
`having 12 panels
`chromosome 12 labeled; and
`[0038] FIG. 4 is a graph showing results from experiments
`done using digital analysis of mixed normal and trisomic
`trisomy 21) DNA.
`(Down Syndrome,
`
`DETAILED DESCRIPTION OF THE
`PREFERRED EMBODIMENT
`
`Outline
`[0039] I. Overview
`[0040] II. Description of Steps
`[0041] A. Tissue Preparation
`[0042] B. Distribution of DNA molecules
`[0043] C. Detection and Quantification
`[0044] 1. Digital PCR Methods
`[0045] 2. Bead emulsion PCR
`[0046] 3. Microfluidic Dilution with PCR
`[0047] 4. Single molecule detection and/or sequenc-
`ing
`
`[0048] D. Quantitative Evaluation
`
`[0049] III. Specific applications
`
`[0050] A. Preparation for trisomy with frequency analy-
`sts.
`
`[0051] B Sample Protocol
`
`[0052]
`
`IV. Examples
`
`I. OVERVIEW
`[0053] The methods and materials described below apply
`for analyzing numerous nucleic acids contained
`techniques
`in a tissue sample
`serum or, more preferably,
`(preferably
`plasma) containing a mixture of DNA from both the mother
`and the fetus, and allowing detection of small but statisti-
`cally significant diflerences.
`the analysis of
`[0054] The present
`involves
`invention
`maternal blood for a genetic condition, wherein
`the mixed
`fetal and maternal DNA in the maternal blood is analyzed
`to
`distinguish a fetal mutation or genetic abnormality
`from the
`background of the maternal DNA. It has been found that,
`using a combination of steps, a DNA sample containing
`DNA from both the mother and the fetus can be analyzed
`to
`distinguish a genetic condition present in a minor fraction of
`the fetal DNA. The method
`the DNA, which represents
`employs "digital analysis, " in which the DNA in the sample
`is isolated to a nominal
`single target molecule
`in a small
`reaction volume. Each sample mixture has a possibility of
`in it less than I target (ke. , 0 target) or
`having distributed
`more than one target. Next, the target molecules are detected
`in each reaction well, preferably as target sequences which
`are amplified, which may include a quantization of starting
`copy number of the target sequence,
`that is, 0, I, 2, 3, etc.
`A control
`to distinguish
`is used
`an abnormal
`sequence
`increase in the target sequence, e. g. , a trisonomy. Thus there
`is a difl'erential detection of target sequences, one of which
`is chosen to represent a normal genotype present
`in both
`and one of which
`is chosen for
`mother and ofl'spring,
`detection of an abnormal genotype
`in the ofl'spring, where
`in the ofl'spring will be diflerent
`from
`the target sequence
`that of the mother, e. g. in trisomy.
`[0055] FIG. 1A illustrates an embodiment where quanti-
`tative detection, e. g. quantitative
`real time PCR, is used.
`Blood 10 is processed to obtain plasma DNA 12, which is
`into aliquots 14. These are added to
`diluted and distributed
`IA through 5D. Shown
`in the wells are
`reactions wells
`targets representing chromosomes 21 and 22. In well 2A, no
`target DNA is found; some wells (not shown) may have
`excess DNA. In well 3B, fetal DNA having
`trisomy 21
`is found. The remainder of the wells
`(Down Syndrome)
`contains maternal DNA. The DNA
`is amplified
`and/or
`is obtained, as shown at
`labeled and a quantitative
`readout
`16. Peak 18 representing well 3B will be 50% higher
`than
`the peaks from the other well, or the peaks from a reference
`sequence on chromosome 22. Well A2, lacking either 21 or
`22, will have no peak. The peaks are shown at 20. A single
`such as wells
`run will have numerous
`random variations,
`that have no target sequence, or have duplication
`through
`sample variability. Also, samples with no target will clearly
`result in no peak at all; wells with two or more targets, will
`give peaks significantly higher than peak 18, i. e. , 2x or 2. 5x
`controls. These results are distinguished by running a mul-
`titude of reactions, followed by statistical analysis
`that can
`from true results.
`discriminate
`random variations
`
`Ariosa Exhibit 1006, p. 10
`
`

`

`US 2007/0202525 A1
`
`Aug. 30, 2007
`
`[0056] FIG. 1C illustrates an embodiment where the DNA
`in a more dilute fashion (less than I, or about
`is distributed
`one half genome equivalents per well). In this case chro-
`mosome 21 labels (primers) will generate more positives
`than chromosome 22 (a diploid chromosome) specific labels
`(e. g. , primers) due simply to the slightly greater abundance
`of chromosome
`21 in a trisomy-containing
`sample. As
`shown, some wells will contain positives 20 for both chro-
`mosomes, some will contain negatives 22 for both chromo-
`somes, but some will contain blanks 24 for the diploid
`chromosome and peaks for the trisomic chromosome, due to
`its greater abundance. The data from a higher peak 18 is not
`used in this mode. As explained below, this slight difl'erence
`can be made statistically
`significant by examining a large
`number of wells, and by the sensitivity of the present method
`to a single molecule.
`
`the
`
`[0057] Thus, the present method comprises generally
`following steps:
`[0058] 1. Obtaining
`a tissue containing DNA from a
`to have about 3%
`subject, which DNA is known
`pregnant
`fetal DNA. This material
`is preferably drawn blood, and the
`circulating DNA is found in the blood plasma, rather than in
`cells. The blood or plasma may optionally be enriched for
`to
`fetal DNA by known methods, such as size fractionation
`less than about 300 bp. Alterna-
`select for DNA fragments
`tively, maternal DNA, which tends to be larger than about
`500 bp may be excluded. Another enrichment
`step may be
`to treat the blood sample with formaldehyde,
`as described in
`Dhallan et al. "Methods to Increase the Percentage of Free
`Fetal DNA Recovered From the Maternal Circulation, " J.
`Am. Med. Soc. 291(9): 1114-1119 (March 2004).
`[0059] 2. Distributing
`single DNA molecules
`from this
`sample to a number of discrete reaction samples, where the
`number of reaction samples
`is selected to give a statistically
`significant result for the number of copies of a target in the
`the reaction sample is confined to
`DNA molecules. Further,
`into close
`to bring the reaction molecules
`a small volume
`approximation. The amount of DNA molecule per reaction
`on the order of one copy of the
`is preferably
`sample
`chromosome of interest equivalent per reaction sample.
`[0060] 3. Detecting the presence of the target in