USOO7888017BZ
`
`(12) United States Patent
`
`Quake et a1.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 7,888,017 B2
`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
`11.S.C. 154(1)) by 65 days.
`
`(21) App1.No.: 11/701,686
`
`(22)
`
`Filed:
`
`Feb. 2. 2007
`
`(65)
`
`Prior Publication Data
`
`US 2007/0202525 Al
`
`Aug. 30. 2007
`
`Related US. Application Data
`
`(60) Provisional application No. 60/764.420. filed on Feb.
`2. 2006.
`
`(51)
`
`Int. Cl.
`(2006.01)
`CIZQ [/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] file for complete search history.
`
`(56)
`
`References Cited
`U .S. PATENT DOCUMENTS
`
`5,641,628 A
`5.879.883 A "
`6.143.496 A
`6.258.540 Bl
`6.440.706 131
`6.927.028 B2
`7.476.363 132
`200310022207 Al "'
`20030186255 A] "'
`20040137470 A1“
`20040203037 Al
`20050019792 A1
`20050129581 Al
`20050145496 A]
`20050221341 A]
`20050221373 Al
`20050252773 A1
`20060252068 A]
`20060252071 A]
`20070059680 Al
`20070212689 A]
`200750238105 A]
`20080026390 Al
`20080050739 A]
`20080070792 A1
`20080138809 A]
`200810153090 A]
`2008018226] A]
`20080193927 A1
`
`................. 435.96
`
`651997 Bianchi
`351999 Benson et a1.
`11/2000 Brown ctal.
`T2001 1.0 eta].
`82002 Vogelstein eta].
`8.2005 1.0 el al.
`1.2009 Ungcret al.
`4356
`152003 Balasubmmanian ct a].
`1052003 Williams et al.
`............... 4356
`.. 4356
`7:2004 Dhallan
`
`102004 Lo et 3].
`132005 McBrideet al.
`6‘ 2005 McBride cl al.
`72005 Goodsaid eta].
`10200.5 Shimkcts el al.
`10.2005 Enzelbergeret a1.
`11‘2005 McBride et al.
`1132006 1.0 eta].
`“2006 1.0 el 3].
`352007 Kapur et a1.
`92007 Bianchi ct al.
`1052007 Barrett et a1.
`1.2008 Stoughlon eta].
`22008 Stoughton eta].
`3.42008 Stoughton ct al.
`62008 Kapuret al.
`62008 L0 eta].
`7.92008 Bianchi
`892008 Mann et al.
`
`200890213775 A1
`200890220422 A]
`20000029377 A1
`200910087847 A1
`201090216151 A1
`201090216153 A1
`
`9/2008 Brody et a1.
`92008 Shoemakcretal.
`[2009 1.0 end.
`42009 1.0 eta].
`82010 Lapidusetal.
`8v‘2010 Lapiduset a1.
`
`FOREIGN PATENT DOCUMENTS
`
`151’
`WO
`WO
`WO
`WO
`WO
`W()
`
`0994 963 81
`03048295 A]
`2006010610 A2
`2007044091 A2
`2009013492 A]
`2009013496 A]
`2009019455 AZ
`
`512003
`62003
`252006
`432007
`112009
`]»’2009
`22009
`
`OTHER PUBLICATIONS
`
`Pohl et a1. Expert Reviews in Molecular Diagnosis. 2004. 4: 4 1-47.‘
`(‘han et al. (‘linica1(‘hemistry. 2004. 50: 88-92“
`Nelson et a1(Var Sanguinis. 2001. 80: 112-116.‘
`Maloncy ct a]. Journal Clinical Investigation. 1999. 104: 41—47.‘
`Maureen Martin. et al.. “A Method for Using Serum or Plasma as a
`Source 01' DNA for 111A Typing." Human Immunology. 1992. vol.
`33. 108-113.
`Stuan L. Emanuel. et al.. “Amplification of Specific Gene Products
`from Human Sentm." 6.4724. 1993. vol. 10. No. 6. 144-146.
`Y-M. 1). Lo. et al.. “Prenatal Sex Determination by DNA Amplifica-
`tion from Maternal Peripheral Blood."T/u- Lancet. Dec. 9. 1989.
`1363-1365.
`Y-M. D. Lo. et al.. “Detection of fetal RhD sequence from peripheral
`blood of sensitized RhD-negalive pregnant women.” British Journal
`ofHaematology. 1994. vol. 87. 658-660.
`Y. M. Dennis Lo. et al.. "Presence of fetal DNA in maternal plasma
`and sentm." The Lancer. Aug. 16. 1997. vol. 350. 485—487.
`
`(Continued)
`
`Primary Examinerinrla Myers
`(74) Allormfv. 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 PCR 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 villi sampling. Digital amplification allows
`the detection 01' ancuploidy using massively parallel amplifi-
`cation and detection methods. examining. e.g.. 10.000
`genome equivalents.
`
`28 Claims. 5 Drawing Sheets
`
`Page 1 of 25
`
`SEQUENOM EXHIBIT 1008
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`SEQUENOM EXHIBIT 1008
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`Page 1 of 25
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`

`US 7,888,017 BZ
`Page 2
`
`OTHER PUBLICATIONS
`
`Jouni Uitto. et al.. “Probing the fetal genome: progress in non-
`invasive prenatal diagnosis." Ti'endx in Molecular Medicine. Aug.
`2003. vol. 9. No. 8. 339-343.
`Sinuhe Hahn. et al.. “Prenatal Diagnosis Using Fetal Cells and Cell-
`Free Fetal DNA in Maternal Blood: What is Currently Feasible?"
`Clinical Obstetrics and Gynecology. Sep. 2002. vol. 45. No. 3. 649-
`656.
`Barbara Pertl. et al.. “Fetal DNA in Maternal Plasma: Emerging
`Clinical Applications." Obstetrics and Gynecology. Sep. 2001. vol.
`98. No. 3. 483-490.
`Leo LM. Poon. et al.. “Circulating fetal DNA in maternal plasma.”
`Clinical Chimica Acta. 2001. vol. 313. 151-155.
`Y-M. D. Lo. et al.. “Fetal DNA in Maternal Plasma." Ann. N. If Acad.
`Sci. Apr. 2000. vol. 906. 141-147.
`Y-M. D. Lo. ct al.. “Detection of single-copy fetal DNA sequence
`from maternal blood." The Lancet. Jun. 16. 1990. vol. 335. 1463-
`1464.
`Y.M. Dennis Lo. et al.. “Quantitative Analysis of Fetal NA in Mater-
`nal Plasma and Sentm: Implications for Noninvasive Prenatal Diag-
`nosis” Am J. Hum. Gene!.. 1998. vol. 62. 768-775.
`Bert Vogelstein. ct al.. “Digital PCR." Proc. Natl. Acad. Sci. USA.
`Aug. 1999. vol. 96.. 9236-9241.
`Rossa W.K. Chiu. et al.. “Effects of Blood-Processing Protocols on
`Fetal and Total DNA Quantification in Maternal Plasma." Clinical
`Chemistry. 2001. vol. 47. No. 9. 1607-1613.
`Ilona Hromadnikova. et al.. “Quantitative analysis of DNA levels in
`maternal plasma in normal and Down syndrome pregnancies." Bio
`Med Central. May 2002. 1-5.
`Enders KO. Ng. et al.. “The Concentration of Circulating
`Conicotropin-releasing Hormone mRNA in Maternal Plasma Is
`Increased in Preeclampsia." Clinical Chemisllj'. 2003. vol. 49. No. 5.
`727-731.
`ldo Braslavsky. et al.. “Sequence information can be obtained from
`single DNA molecules." PNAS. Apr. 2003. vol. 100. No. 7. 3960-
`3964.
`Jun Zhu. et al.. “Single Molecule Profiling ofAlternative Pre-mRNA
`Splicing.” Science. Aug. 2003. vol. 301. 836-838.
`Devin Dressman. et al.. “Transforming single DNA molecules into
`fluorescent magnetic particles for detection and enumeration of
`genetic variations." PNAS. Jul. 2003. vol. 100. No. 15. 8817-8822.
`Eugene Y. Chan. et al.. “DNA Mapping Using Microtluidic Stretch-
`ing and Single-Molecule Detection of Fluorescent Site-Specific
`Tags.” Genome Research. 2004. vol. 14. 1 137-1146.
`Al Sheng Xiong. ct al.. “A simple. rapid. high-fidelity and cost-
`efl‘ective PCR-based two-step DNA synthesis method for long gene
`sequences." NucleicAcidx Research. Apr. 19. 2004. vol. 32. No. 12.
`c98.
`v
`Jong Wook Hong. eta1.. “Molecular biology on a microfluidic chip.‘
`Journal ofPlnwics: Condensed Matter. 2006. vol. 18. $691-$701.
`Elimbeth A. Ottesen. et al.. “Microfiuidic Digital PCR Enables
`Multigene Analysis of Individual Environmental Bacteria." Science.
`Dec. 2006. vol. 314. 1464-1467.
`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.. “Microtluidic Single-Cell mRNA Isolation
`and Analysis." American Chemical Society. Mar. 2006. p. A-F.
`Ryo Kimura. et al.. “The DYRK 1 A gene. encoded in chromosome 21
`Down syndrome critical region. bridges between fi-amyloid produc-
`tion and tau phosphorylation in Airheimer disease." Human Molecu-
`lar Genetics. Nov. 29. 2006. vol. 16. No. 1. 15-23.
`Y. M. Dennis Lo. eta1.. “Prenatal diagnosis: progress through plasma
`nucleic acids.” Nature. Jan. 2007. vol. 8. 71-76.
`
`Tetsuya S. Tanaka. et al.. “Genome-wide expression profiling of
`mid-gestation placenta and embryo using a 15.000 mouse develop-
`mental cDNA microan‘ay." PNAS. Aug. 2000. vol. 97. No. 16. 9127-
`9132.
`“Separation of RNA & DNA by Gel Filtration Chromatography.”
`Edvorek. 1987. 1-9.
`International Search Repon and Written Opinion for PCT/US2007/
`003209. received Sep. 22. 2008.
`Fiona M. F. Lun. ct al.. “Microtluidics Digital PCR Reveals a Higher
`than Expected Fraction of Fetal DNA in Maternal Plasma." Clinical
`Chemistry. 2008. vol. 54. No. 10. 1664-1672.
`Young 110 Yang. et al.. “Rapid Prenatal Diagnosis of Trisomy 21 by
`Real-time Quantitative Polymerase Chain Reaction with Amplifica-
`tion of Small Tandem Repeats and SIOOB in Chromosome 21."
`Yonsei Medical Journal. 2005. vol. 46. No. 2. 193-197.
`Vincenzo Cirigliano. et al.. “Clinical application of multiplex quan-
`titative fluorescent polymerase chain reaction (QF-PCR) for the rapid
`prenatal detection of common chromosome aneuploidies." Molecu-
`lar Human Reproduction. 2001. vol. 7. No. 10. 1001-1006.
`Rebocca Sparkcs. et al.. “New Molecular Techniques for the Prenatal
`Detection ofChromosomal Aneuploidy.” JOGC. Jul. 2008. No. 210.
`617-621.
`Y.M. Dennis Lo. et al.. "Digital PCR for the molecular detection of
`fetal chromosomal aneuploidy.” PNAS. Aug. 7. 2007. vol. 104. No.
`32.13116-13121.
`Haissam Rahil. et al.. Rapid detection of common autosomal
`aneuploidies by quantitative fluorescent PCR on uncultured
`amniocytes. European Journal of Human Genetics. 2002. vol. 10.
`462-466.
`Bernhard Zimmermann. "Molecular Diagnosis in Prenatal Medi-
`cine." PhD. Thesis. 2004. Only Chapter I (Introduction). pp. 1-19.
`Yuk-Ming Dennis Lo. “Noninvasive prenatal detection of fetal chro-
`mosomal aneuploidies by maternal plasma nucleic acid analysis: a
`reviewofthecurrent state ofthean." BJOG. 2009. vol. 116. 152-157.
`Jay Shendure. et al.. "Next-generation DNA sequencing." Nature.
`2008. vol. 26. No. 10. 1135-1145,
`11. Christina Fan. et al.. "Noninvasive diagnosis of fetal aneuploidy
`by shotgun sequencing DNA from maternal blood." PNAS. Oct. 21.
`2008.Vol. 105. 16266-16271.
`Richard A. White 111. et al.. “Digital PCR provides sensitive and
`absolute calibration for high throughput sequencing." BMC Genom-
`ics. Mar. 19. 2009. 10:116.
`Frank Diehl. et al.. "Digital quantification of mutant DNA in cancer
`patients." Curr Opin Oncol. 2007. 19:36-42.
`1).]. Sykes. etal.. "QuantitationofTargets for PCRby UseofLimiting
`Dilution." BioTechniques. 1992. vol. 13. No. 3. 444-449.
`B. Zinuncrman. et al.. "Novel Real-Time Quantitative PCR Test for
`Trisorny 21." Jan. 1. 2002. Clinical Chemistry. American Association
`for Clinical Chemistry. vol. 48. No. 2. 362-363.
`Ying Li. et al.. “Size Separation of Circulatory DNA in Maternal
`Plasma Permits Ready Detection of Fetal DNA Polymorphisms."
`2004. Clinical Chemistry. vol. 50. No. 6. 1002-1001.
`11. Christina Fan. ct al.. “Detection of Aneuploidy with Digital
`Polymerase Chain Reaction." Analytical Chemistry. Oct. 1. 2007.
`vol. 79. No. 19. 7576-7579.
`H. Christina Fan. et al.. "Microlluidic digital PCR enables rapid
`prenatal diagnosis of fetal aneuploidy." American Journal ol'Obstet-
`rics & Gynecology. May 2009. 543e1-543-e7.
`EPO Search Report. 07 763 674.4. Jul. 31. 2009.
`Leutwyler. K.. Mapping Chromosomes 21. Scientific American. May
`15. 2000.
`Tettelin. T.. et al.. “The nucleotide sequence of Saccharomyces
`ccrevisiae chromosome V11.“ Nature 1997. 387:81-84.
`Zavala. A.. ct al.. “Genomic GC content prediction in prokaryotes
`from a sample of genes." Gene 2005. 357(2): 127-143.
`International Search Report. International Application No. PCT/US
`09/‘57136. Mar. 16. 2010.
`
`‘ cited by examiner
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`US. Patent
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`Feb. 15, 2011
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`Sheet 1 of5
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`US 7,888,017 82
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`5
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`US 7,888,017 82
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`1—54
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`US 7.888017 32
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`Chr21 FAM
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`Fig. 2
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`U.S. Patent
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`US 7,888,017 82
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`(stdev)
`ConfidenceInterval
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`Percent Down’s DNA
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`Fig. 4
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`US 7,888,017 32
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`l
`NON-[WASIVE FETAL GENETIC
`
`
`SCREENING BY DIGITAL ANALYSIS
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`This application claims priority from U.S. 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 U.S. Government support
`under contract 0535870 awarded by the National Science
`Foundation. The U.S. (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/1FC.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
`
`4C]
`
`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 head contains one
`amplicon ofdigital PCR. (Dressman et a1. 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.“ U.S. 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 maternal 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'ec 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 matemal 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
`
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`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 ofPCR
`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 chorionic 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 1 2 to
`the amount of a gene located on another autosomal chromo-
`some. Ifthe ratio ofthese two genes is 1:1. 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 [Terent 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 matemal 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-H l 9
`locus. which is maintained in a methylated DNA status in the
`
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`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. 'lhe 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 SUM MARY 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 il'the 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
`
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`a matcmal 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 perfomred 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 21. the
`mixture may be diluted such that. on average. one may detect
`two chromosomes present in a maternal DNA. and three
`chromosomes in a Down Syndrome fetal DNA. Altematively.
`the method may be perfonned 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 maternal 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. ln 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.
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`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 0 1‘genetic 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 a known genetic disorder in the fetal genetic mate-
`rial not present in the maternal genetic material. For example
`chromosome trisomies may include partial. mosaic. ring. 18.
`l4. l3. 8. 6. 4 etc. A listing of known abnormalities may be
`found in the OMIM Morbid map. at world wide web address
`ncbi.n1m.nih.gov/Omim/getmorbid.cgi.
`In gener