(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`(43) International Publication Date
`12 June 2008 (12.06.2008)
`
`(51) International Patent Classification:
`CIZQ 1/68 (2006.01)
`
`i
`
`.. ‘
`
` (10) International Publication Number
`
`WO 2008/070862 A2
`
`(74) Agents: MIRBIIRA, Suman et a1; Cooley Godward Kro—
`nish LLP, 777 6th Street, NW, Suite 1100, Washington, DC
`20001 (US).
`
`(81) Designated States (unless otherwise indicated, for evety
`kind of national protection available): AE, AG, AL, AM,
`AT, AU, AZ, BA, BB, BG, BII, BR, BW, BY, BZ, CA, CII,
`CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, EG,
`ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL,
`IN, IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK,
`LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, MW,
`MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PG, PH, PL,
`PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, SV, SY,
`TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA,
`ZM, ZW.
`
`(84) Designated States (unless otherwise indicated for evety
`kind of regional protection available): ARIPO (BW, GII,
`GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FT,
`FR, GB, GR, HU, IE, IS, IT, LT, LU, LV, MC, MT, NL, PL,
`PT, RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, CI, CM,
`GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG).
`Published:
`
`without international search report and to be republished
`upon receipt of that report
`
`(21) International Application Number:
`PCT/U82007/086862
`
`(22) International Filing Date:
`7 December 2007 (07.12.2007)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`60/869,090
`
`7 December 2006 (07.12.2006)
`
`US
`
`(71) Applicant (for all designated States except US}: BIO-
`CEPT, INC. [US/US]; 5810 Nancy Ridge Drive, Suite
`150, San Diego, CA 92121 (US).
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): BHATT, Ram
`[US/US]; 3575 Santoro Way, San Diego, CA 92130
`(US). FAN, Wen-Hua [US/US]; 11181 Kelowna Road,
`San Diego, CA 92126 (US). TIM, Roger
`[US/US];
`8268 Gilman Drive, Unit 7, La Jolla, CA 92037 (US).
`BISCHOFF, Farideh [US/US]; 3314 N. Briarpark Lane,
`Sugar Land, TX 77479 (US).
`
`(54) Title: NON—INVASIVE PRENATAL GENETIC SCREEN
`
`
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`W02008/070862A2|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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`
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`(57) Abstract: The present invention provides methods and kits useful for genetic testing or screening of fetuses using nucleic acid
`samples isolated from cervical mucus samples of fetus hosts.
`
`

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`WO 2008/070862
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`PCT/US2007/086862
`
`NON—INVASIVE PRENATAL GENETIC SCREEN
`
`FIELD OF THE INVENTION
`
`LII
`
`10
`
`[0001]
`
`This invention relates generally to the isolation of fetal nucleic acid and
`
`prenatal screening or testing of genetic and chromosomal abnormalities.
`
`BACKGROUND OF THE INVENTION
`
`[0002]
`
`Prenatal testing or screening is usually performed to determine the gender
`
`of the fetus or to detect genetic disorders and/or chromosomal abnormalities in the fetus
`
`during pregnancy. As of today, over 4000 genetic disorders, caused by one or more faulty
`
`genes, have been recognized. Some examples include Cystic Fibrosis, Huntington’s Disease,
`
`Beta Thalassaemia, Myotonic Dystrophy, Sickle Cell Anemia, Porphyria, and Fragile—X—
`
`Syndrome. Chromosomal abnormality is caused by aberrations in chromosome numbers,
`
`duplication or absence of chromosomal material, and by defects in chromosome structure.
`
`Some examples of chromosomal abnormalities are trisomies, namely trisomy 16 , a major
`
`cause of miscarriage in the first trimester, trisomy 21 (Down syndrome), trisomy 13 (Patau
`
`syndrome), trisomy l8 (Edwards syndrome), Klinefelter’s syndrome (47, XXY), (47, XYY),
`
`and (47, XXX); the absence of chromosomes (monosomy), cg, Turner syndrome (45, X0);
`
`chromosomal translocations, deletions and/or microdeletions, e.g., Robertsonian
`
`translocation, Angelman syndrome, DiGeorge syndrome and Wolf-Hirschhorn Syndrome.
`
`[0003']
`
`Currently available prenatal genetic tests usually involve invasive
`
`procedures. For example, chorionic villous sampling (CVS) performed on a pregnant woman
`
`around 10—12 weeks into the pregnancy and amniocentesis performed at around l4-l6 weeks
`
`all contain invasive procedures to obtain the sample for testing chromosomal abnormalities in
`
`a fetus. Fetal cells obtained via these sampling procedures are usually tested for
`
`chromosomal abnormalities using cytogenctic or fluorescent in situ hybridization (FlSH)
`
`k) VI
`
`analyses.
`
`[0004}
`
`While these procedures can be useful for detecting chromosomal
`
`aberrations, they have been shown to be associated with the risk of miscarriage. Therefore
`
`amniocentesis or CVS is only offered to women perceived to be at increased risk, including
`
`those of advanced maternal age (>35 years), those with abnormal maternal serum screening
`
`30
`
`or those who have had a previous fetal chromosomal abnormality. As a result of these tests
`
`the percentage ofwomen over the age of 35 who give birth to babies with chromosomal
`
`l
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`‘JI
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`
`aberrations such as Down syndrome has drastically reduced. However, lack of appropriate or
`
`relatively safe prenatal testing or screening for the majority of pregnant women has resulted
`
`in about 80% ofDown syndrome babies born to women under 35 years of age.
`
`[0005]
`
`Thus there is a need for diagnostic screening tests for the general
`
`population of pregnant women, especially tests directed to identifying fetal chromosomal
`
`aberrations as well as other genetic variations or defects.
`
`SUMMARY OF THE INVENTION
`
`[0006]
`
`The present invention is based, in part, on the discovery that cervical
`
`mucous is a good natural reservoir for migrated placental cells, e. g., fetal cells as well as for
`
`isolating fetal nucleic acids. Accordingly the present invention provides methods and kits
`
`useful for testing or screening for genetic abnormalities in fetuses using fetal nucleic acids
`
`isolated from cervical mucus samples.
`
`In addition, the present invention provides primers
`
`and probes useful for nucleic acid amplification of, ag, genetic markers, especially using
`
`relatively small size amplicons in fetal genetic screening.
`
`[0007]
`
`In one embodiment of the invention, it provides a method for conducting a
`
`genetic test of a fetus. The method comprises isolating a nucleic acid sample from a cervical
`
`mucus sample obtained from a female subject containing the fetus, wherein the nucleic acid
`
`sample consists essentially of polynucleotides in a size ranging from about 50 base pairs to
`
`about 300 base pairs and wherein the result of a genetic test on the nucleic acid sample is
`
`indicative of a genetic composition of the fetus.
`
`[0008]
`
`In another embodiment of the invention, it provides a method of isolating a
`
`fetal nucleic acid sample. The method comprises isolating a nucleic acid sample consisting
`
`essentially of polynucleotides of about 50 base pairs to about 300 base pairs in length from a
`
`cervical mucus sample obtained from a female subject containing the fetus.
`
`[0009]
`
`In yet another embodiment of the invention, it provides a genetic testing
`
`kit suitable for testing the genetic composition of a fetus. The kit comprises a pair of primers
`
`suitable for amplifying a desired allele or genetic marker, wherein the amplified nucleotide
`
`fragment is less than about 200 base pairs and wherein the desired allele is not uniquely
`
`associated with the Y chromosome. In other embodiments, the kit comprises an isolated
`
`DNA sample from a cervical mucus sample obtained from a female subject containing the
`
`fetus. The DNA sample consists essentially of polynucleotides in a size ranging from about
`
`50 base pairs to about 200 base pairs.
`
`l‘x)
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`WO 2008/070862
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`PCT/U52007/086862
`
`{0010]
`
`In still another embodiment of the invention, it provides an isolated DNA
`
`sample useful for genetic testing of a fetus. The DNA sample can be obtained by isolating
`
`DNA fragments in a size ranging from about 50 base pairs to about 200 base pairs from a
`
`cervical mucus sample obtained from a female subject containing the fetus.
`
`BRIEF DESCRIPTION OF THE FIGURES
`
`{0011]
`
`F 1G. 1 shows the size fractionation of total DNA obtained from cervical
`
`mucus on a 10% polyacrylamide gel. “Band A,” corresponding to a polynucleotide length of
`
`around 50-200 base pairs contains fetal DNA.
`
`[0012]
`
`FIG. 2 shows an example of PCR electropherogram demonstrating that in
`
`10
`
`one experiment the fetal signals match between fetal tissue DNA and the 50—200 bp fragment
`
`of DNA isolated from a cervical mucus sample.
`
`[0013]
`
`FIG. 3 shows another example of PCR electropherogram demonstrating
`
`that in another experiment the fetal signals match between fetal tissue DNA and the 50-200
`
`bp fragment of DNA isolated from a cervical mucus sample.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`20
`
`25
`
`{0014}
`
`It is the discovery of the present invention that cervical mucus samples can
`
`,
`be a great source for fetal cells as well as fetal nucleic acids. Accordingly, the present
`invention provides methods, reagents and kits useful for testing or screening fetus for genetic
`
`abnormalities using nucleic acids isolated from cervical mucus samples.
`
`[0015]
`
`In addition, the present invention provides primers and probes useful for
`
`nucleic acid amplification, eg, of genetic markers, especially using relatively small size
`
`amplicons in fetal genetic screening.
`
`{0016]
`
`According to one aspect of the present invention, it provides methods for
`
`conducting genetic tests of a fetus by isolating one or more nucleic acid samples from one or
`
`more cervical mucus samples obtained from a female subject containing the fetus.
`
`In generalr
`
`the nucleic acid sample useful for the methods of the present invention can be a DNA sample
`
`RNA sample: or a combination thereof including any DNA, eDNAg or RNA derived from
`
`one or more nucleic acid samples isolated from one or more cervical mucus samples.
`
`{(3037}
`
`in one embodiment, the nucleic acid sample useful for the methods of the
`
`present invention is a DNA sample. In another embodiment: the nucleic acid sample useful
`
`for the methods of the present intention is substantially free of proteins or polypeptides. In
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`

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`WO 2008/070862
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`PCT/U52007/086862
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`Ur
`
`10
`
`15
`
`20
`
`25
`
`30
`
`yet another embodiment, the nucleic acid sample useful for the methods of the present
`
`invention is isolated by any known or later discovered size fractionation method including,
`
`but not limited to, gel electrophoresis, capillary electrophoresis, size exclusion matrixes, and
`
`size fractionation columns.
`
`[0018]
`
`in still another embodiment, the nucleic acid sample useful for the
`
`methods of the present invention is in a size range representative of, or substantially
`
`associated, with fetal nucleic acid.
`
`In still another embodiment, the nucleic acid sample
`
`useful for the methods of the present invention is in a size range substantially free of nucleic
`
`acid from the host of the fetus. For example, the nucleic acid sample useful for the methods
`
`of the present invention can be in a size range from about 50 to about 1000 base pairs, from
`
`about 50 to about 500 base pairs, from about 50 to about 400 base pairs, from about 50 to
`
`about 300 base pairs, from about 50 to about 250 base pairs, from about 50 to about 200 base
`
`pairs, from about 50 to about 150 base pairs, or from about 50 to about 100 base pairs or a
`
`combination thereof, and optionally, does not contain a substantial amount, e.g., more than
`
`0.5%, 1%, 2%, 3%, 4%, or 5% of nucleic acids from any other size range or source.
`
`[0019]
`
`According to the present invention, the nucleic acid sample useful for the
`
`methods of the present invention can be isolated from a cervical mucus sample from the host
`
`of a fetus, e.g., a pregnant woman. The cervical mucus sample ofthc present invention can
`
`be obtained from the host of a fetus, at any time during the pregnancy, for example. during
`
`the first or second trimester, by any means now known or later discovered in the art.
`
`In
`
`general, a cervical mucus sample, e.g., an endocervical mucus sample, can be obtained using
`
`techniques such as transcervical swabs, endocervical lavage, scrapes, cytobrush, aspiration,
`
`intrauterine lavage, or a combination thereof,
`
`[0020]
`
`In one embodiment, the cervical mucus sample of the present invention is
`
`a fresh sample, e.g, without substantial preservation or processing. In another embodiment,
`
`the cervical mucus sample is a sample preserved from a fresh sample, e.g., preserved in a
`
`suitable aqueous preservation or transportation medium, or alternatively, a sample of a
`
`medium containing nucleic acids leached from one or more cervical mucus samples. Without
`
`being bound to any theory, it is believed that nucleic acid will diffuse out from the cervical
`
`mucus into a fluid that is in contact with the mucus. Fetal nucleic acid will thus be present
`
`both in the cervical mucus sample as well as in the media in which the sample is stored
`
`andfer transported. Accordingly. the nucleic acid sample useful for the methods of the
`
`present invention can be obtained directly from the cervical mucus sample, or from the
`
`medium, for example, preservation medium, transportation medium, or any aqueous medium.
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`PCT/U52007/086862
`
`that is in contact with the cervical mucus. Examples of transportation media include, but are
`
`not limited to, any tissue culture medium known to one of skill in the art, e.g., RPMI-1640
`
`medium. In yet another embodiment, the cervical mucus sample of the present invention is
`
`maintained or stored between about 40C and about 20°C, e.g., in a low calcium basal
`
`U:
`
`medium.
`
`[0021]
`
`In still another embodiment, the cervical mucus sample of the present
`
`invention is a treated sample, e. g, a fresh sample or preserved sample treated with any
`
`suitable reagent(s) to facilitate mucous dissolution which in turn, assists in isolation of
`
`nucleic acid components from the sample. For example, the cervical mucus sample can be a
`
`10
`
`sample treated with mucolytic agent(s) or mucinase(s), e.g., N-acetyl-L»cysteine, L—cysteine,
`
`dithiothreitol (DTT), brornhexine hydrochloride, and any ofthe hyaluronidases, including
`
`hyaluronate lyase, hyaluronoglucosarninidase. and hyaluronglucuronidase.
`
`In another
`
`example, the cervical mucus sample of the present invention is a sample treated with
`
`enzyme(s), e.g., sugar hydrolysis enzyme(s) such as B-galactosidase or invertase, or
`
`proteinase, or pepsin or combinations thereof. The cervical mucus sample may also be
`
`treated with chemicals known in the art to induce apoptosis to release fetal nucleic acid.
`
`[0022]
`
`In another embodiment, the cervical mucus sample of the present
`
`invention is a sample treated to enrich fetal nucleic acid and/or reduce maternal nucleic acid
`
`content. For example, the cervical mucus sample can be treated to reduce or degrade any
`
`nucleic acid, e.g., DNA that is characteristic of maternal DNA. One of such nucleic acid is
`
`hypermethylated maternal DNA. Any means to reduce, degrade, or selectively remove
`
`hypermethylated maternal DNA can be used including, Without any limitation, methylation
`
`specific restriction enzymes such as MchC (BioLabs), antibodies specific for
`
`hypermethylated maternal DNA such as anti—5’—methyl-cytosine antibodies andx’or anti—
`
`methlepG binding protein—2 (MeCPE) antibodies, or ligands or proteins such as MeCP2 that
`
`specifically bind methylated CpG islands in maternal DNA.
`
`[0023]
`
`Alternatively fetal nucleic acid can be enriched using markers specific for
`
`fetal nucleic acids. For example, hypomethylated maspin DNA can be used as a marker for
`
`fetal DNA. In one instance, one can treat total cervical mucous DNA with sodium bisulfite,
`
`which can induce chemical changes in the hypomethylated fetal DNA whereby unmethylated
`
`cytosine of fetal DNA is converted into uridine (U). Such change can be used to
`
`preferentialiy isolate or enrich fetal DNA, e.g., to preferentiaily amplify fetal DNA
`
`containing uridine(s) converted from cytosine(s).
`
`20
`
`h) UI
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`WO 2008/070862
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`PCT/U52007/086862
`
`[0024]
`
`According to the present invention, the nucleic acid sample of the present
`
`invention can be used to conduct genetic tests or screening of a fetus. In particular, the
`
`nucleic acid sample of the present invention can be used to test or screen the genetic
`
`composition of a fetus, cg, chromosomal composition, gene composition, or genetic marker
`
`or finger printing pattern of a fetus.
`
`In one embodiment, testing or screening a genetic
`
`composition of a fetus includes probing for chromosomal abnormalities including, without
`
`any limitation, monosomy, partial monosomy, trisomy, partial trisomy. chromosomal
`
`translocation, chromosomal duplication, chromosomal deletion or microdeletion, and
`
`chromosomal inversion.
`
`[0025]
`
`In general, the term “monosomy” refers to the presence of only one
`
`chromosome from a pair of chromosomes. Monosomy is a type of aneuploidy. Partial
`
`monosomy occurs when the long or short arm of a chromosome is missing. Common human
`
`genetic disorders arising from monosomy include: X0. only one X chromosome instead of the
`
`usual two (XX) seen in a normal female (also known as Turner syndrome); cri du chat
`
`syndrome, a partial monosomy caused by a deletion of the end of the short p (from the word
`
`petit, French for small) arm of chromosome 5; and 1p36 Deletion Syndrome, a partial
`
`monosomy caused by a deletion at the end of the short p arm of chromosome 1.
`
`[0026]
`
`In contrast, the term “trisomy” refers to the presence of three, instead of
`
`the normal two, chromosomes of a particular numbered type in an organism. Thus the
`
`presence of an extra chromosome 21 is called trisomy 21. Most trisomies, like most other
`
`abnormalities in chromosome number, result in distinctive birth defects. Many trisomies
`
`result in miscarriage or death at an early age. A partial trisomy occurs when part of an extra
`
`chromosome is attached to one of the other chromosomes, or if one of the chromosomes has
`
`two copies of part of its chromosome. A mosaic trisomy is a condition where extra
`
`chromosomal material exists in only some of the organism’s cells. While a trisomy can occur
`
`with any chromosome, few babies survive to birth with most trisomies. The most common
`
`types that survive without spontaneous abortion in humans include: Trisomy 21 (Down
`
`syndrome); Trisomy l8 (Edwards syndrome); ’l‘risomy l3 (Patau syndrome); Trisomy 9;
`
`Trisomy 8 (Warkany syndrome 2); Trisomy l6 {which is the most common trisomy in
`
`humans, occurring in more than 1% of pregnancies. This condition, however, usually results
`
`in spontaneous miscarriage in the first trimester).
`
`'l‘risomy involving sex chromosomes
`
`include: XXX (Triple X syndrome); XXY (Klinefelter‘s syndrome}; and XYY (XYY
`
`syndrome).
`
`Ur
`
`l0
`
`15
`
`20
`
`h.) (J1
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`30
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`{0027]
`
`In another embodiment, testing or screening a genetic composition of a
`
`fetus includes probing for allele or gene abnormalities, eg, one or more mutations such as
`
`point mutations, insertions, deletions in one or more genes.
`
`[0028]
`
`in yet another embodiment, testing or screening a genetic composition of a
`
`U1
`
`fetus includes probing for one or more polymorphism patterns or genetic markers, e.g., short
`
`tandem repeat sequences (STRS), single nucleotide polymorphisms (SNPs), etc.
`
`{0029]
`
`In still another embodiment, testing or screening a genetic composition of
`
`a fetus includes probing for any genetic abnormality corresponding to or associated with a
`
`condition or disorder, e.g., Cystic Fibrosis, Sickle-Cell Anemia, Phenylketonuria, Tay-Scahs
`
`Disease, Adrenal Hyperplasia, Fanconi Anemia, Spinal Muscularatrophy, Duchenne's
`
`Muscular Dystrophy, Huntington’s Disease, Beta Thalassaemia, Myotonic Dystrophy,
`
`Fragile-X Syndrome, Down Syndrome, Edwards Syndrome, Patau Syndrome, Klinefelter’s
`
`Syndrome, Triple X syndrome, XYY syndrome, Trisomy 8, Trisomy 16, Turner Syndrome,
`
`Robertsonian translocation, Angelman syndrome, DiGeorge Syndrome, Wolf-Hirschhorn
`
`Syndrome, RhD Syndrome, Tuberous Sclerosis, Ataxia Telangieltasia, and Prader—Willi
`
`syndrome,
`
`.
`
`[0030]
`
`In still another embodiment, testing or screening a genetic composition of
`
`a fetus includes probing for any genetic abnormality that is not uniquely associated with Y
`
`chromosome.
`
`[0031]
`
`In still another embodiment, testing or screening a genetic composition of
`
`a fetus includes probing for any genetic condition corresponding to or associated with gender
`
`or paternity of the fetus.
`
`[0032]
`
`Usually genetic tests provided by the present invention use the nucleic acid
`
`sample of the present invention either directly or as templates for “amplification-based”
`
`is.) U:
`
`genetic composition testing assays, including without any limitation, polymerase chain
`
`reaction (“PCR”), real-time polymerase chain reaction (“‘RT-PCR”), Iigase chain reaction
`
`(“LCR”), self-sustained sequence replication (“3SR”) also known as nucleic acid sequence
`
`based amplification (“NASBA”), Q-B‘chlicase amplification, rolling circle amplification
`
`(“RCA"), transcription mediated amplification (“Th/1A”), linker-aided DNA amplification
`
`(/“LADA”; multiple displacement amplification (“MBA”), invader and strand displacement
`
`amplification (“SDA”), Amplification of a nucleotide fragment using a pair of primers
`
`specific for an allele indicates the presence of the allele.
`
`{0033]
`
`in one embodiment, the “amplification-based“ genetic composition testing
`
`assays of the present invention include using primers to generate ampticons less than about
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`WO 2008/070862
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`U)
`
`10
`
`15
`
`200 base pairs, less than about 150 base pairs, or between about 75 to about 150 base pairs.
`
`Exemplary primers of the invention used in the amplification-based assays are provided
`
`herein.
`
`In one embodiment, the primers of the invention include, but are not limited to, the
`
`pairs of primers of SEQ 1D NOS: 1 and 2; SEQ ID NOS: 3 and 4; SEQ 1D NOS: 5 and 6; SEQ
`
`ID NOS: 9 and 10; SEQ ID N05: 11 and 12; and SEQ ID N05: 13 and 14. 1n another
`
`embodiment, exemplary primers ofthe invention include, but are not limited to, the primer
`
`sets listed in Tables 2, 3, 4 and 5.
`
`[0034]
`
`According to another aspect of the present invention, it provides a method
`
`of isolating a fetal nucleic acid sample. The method comprises isolating one or more nucleic
`
`acid samples from a cervical mucus sample obtained from a maternal host of a fetus in a size
`
`range enriched with fetal nucleic acids. Examples of such size range include without any
`
`limitation from about 50 to about 1000 base pairs, from about 50 to about 500 base pairs,
`
`from about 50 to about 400 base pairs, from about 50 to about 300 base pairs, from about 50
`
`to about 250 base pairs, from about 50 to about 200 base pairs, from about 50 to about 150
`
`base pairs, or from about 50 to about 100 base pairs or a combination thereof.
`
`In one
`
`embodiment, the nucleic acid sample does not contain a substantial amount, eg, more than
`
`0.5%, 1%, 2%, 3%, 4%, or 5% of nucleic acids from any other size range or source.
`
`[0035]
`
`According to yet another aspect of the present invention, it provides an
`
`isolated nucleic acid sample useful for genetic testing of a fetus. The nucleic acid sample,
`
`eg, a DNA sample, can be obtained by isolating nucleic acid fragments of from about 50
`
`base pairs to about 100, 200, 300, 400. 500, or 1000 base pairs in length from a cervical
`
`mucus sample obtained from a female subject containing the fetus,
`
`in one embodiment, these
`
`nucleic acid fragments are obtained from the total nucleic acid isolated from the cervical
`
`mucus sample by a size fractionation method. In another embodiment, the isolated nucleic
`
`25
`
`acid is substantially free ofnon-nucleic acid components.
`
`[0036}
`
`According to still another aspect of the present invention, it provides kits
`
`useful for genetic testing or screening of a fetus.
`
`In one embodiment, the kit provided by the
`
`present invention contains one or more pairs of primers useful for genetic composition testing
`
`assays and optionally one or more probes usefui for detecting the amplified product(s) by the
`
`primers.
`
`in another embodiment, the kit provided by the present invention contains one or
`
`more pairs of primers useful for testing one or more polymorphisms or genetic markers of a
`
`fetus. In yet another embodiment, the kit provided by the present invention contains one or
`
`more pairs of primers which are useful for generating amplicons fess than about 200 base
`
`pairs, fess than about $50 base pairs, or between about 75 to about 150 base pairs.
`
`in stilt
`
`

`

`WO 2008/070862
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`PCT/U52007/086862
`
`another embodiment, the kit provided by the present invention contains one or more pairs of
`
`primers for one or more designated chromosomes and the primers are selected from the
`
`primer sets listed in Tables 2, 3, 4 or 5.
`
`In a further embodiment, the kit of the invention
`
`contains the pairs of primers of SEQ ID NOS: I and 2; SEQ ID NOS: 3 and 4; SEQ ID NOS: 5
`
`and 6; SEQ ID NOs: 9 and IO; SEQ ID NOs: II and I2; SEQ ID NOs: 13 and I4; or a
`
`combination thereof. The kit can also optionally contain one or more probes and/or other
`
`suitable reagents useful for detecting the amplified product(s) by the primers. Further, the kit
`
`can comprise instructions for using the pair of primers to test the genetic composition of a
`
`fetus.
`
`[0037]
`
`In still another embodiment, the kit of the present invention contains one
`
`or more nucleic acid samples of the present invention.
`
`In a further embodiment, the kit
`
`provided by the present invention contains the cervical mucus sample of the present invention
`
`and an instruction for isolating the nucleic acid sample of the present invention from the
`
`cervical mucus sample.
`
`EXAMPLES
`
`UI
`
`IO
`
`15
`
`[0038]
`
`The following examples are intended to illustrate but not to limit the
`
`invention in any manner, shape, or form, either explicitly or implicitly. While they are
`
`typical of those that might be used, other procedures, methodologies, or techniques known to
`
`those skilled in the art may alternatively be used.
`
`20
`
`Example 1
`
`[0039]
`
`This Example describes the collection and isolation of fetal DNA from
`
`pregnant women.
`
`[0040]
`
`Cervical mucous samples were collected from patients, after due consent,
`
`by cytobrush method.
`
`In the cytobrush method, a Pap smear cytobrush (6g, MedScand—AB.
`
`Malmo, Sweden) was inserted to a maximum depth of 2 cm and removed while rotating it a
`
`fuli tum (tie, 360°).
`
`In order to remove the transcervical ceIis caught on the brush, the brush
`
`was shaken into a test tube containing 2-3 mi ofa tissue culture medium (etgn RPMI—1640
`
`medium, available ATCC, Virginia) in the presence of 1% Penicillin Streptomycin antibiotic.
`
`In order to concentrate the transeervical cells on microscopic slides eytospin slides were
`
`prepared using eg, a Cytofunnei Chamber Cytocentrifuge (Thermo—Shandon. England}. The
`
`conditions used for cytocentrifugation are dependent on the murkiness of the transeervical
`
`9
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`

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`WO 2008/070862
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`PCT/US2007/086862
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`specimen; if the specimen contained only a few cells, the cells are first centrifuged for five
`
`minutes and then suspended with 1 ml of fresh medium. Once prepared, the cytospin slides
`
`can be kept in 95% alcohol until further use.
`
`U‘I
`
`l0
`
`[0041]
`
`DNA was extracted from fetal tissues, mucous samples or the transport
`
`media using Roche‘s Apoptotic DNA—Ladder Kit following manufacturer’s protocol with
`
`slight modification. Mucous samples were incubated with equal volume of lysis buffer for 30
`
`minutes to 2 hours or until all the mucous had been dissolved. Some samples needed to be
`
`homogenized with a 21 gauze 1.5 inch long needle to facilitate complete mucous dissolution.
`
`Total mucous DNA was then size fractionated on 10% PAGE, also known as 10% TBE gel
`
`(Invitrogen) under non—denaturing conditions, and the small, 100—250 base pair long DNA
`
`band (see Figure l) was sliced out after staining the gel with SYBR Gold stain. Fetal DNA
`
`from the gel was extracted by soaking the crushed gel in 0.3M sodium acetate (pH 5.5) at
`
`37°C for overnight followed by desalting the DNA using Promega’s Wizard SV Genomic
`
`DNA Purification kit.
`
`15
`
`Example 2
`
`[0042]
`
`This Example demonstrates that the DNA obtained from the cervical
`
`mucous samples after PAGE purification is indeed fetal DNA.
`
`[0043]
`
`The total DNA obtained from the cervical swap was size fractionated on
`
`10% PAGE, and the small, 50-250 base pair DNA band (see Figure 1) was sliced out. The
`
`DNA was extracted from PAGE using Promega’s Membrane Binding buffer, and its
`
`concentration was determined by NanoDrop-1000 Spectrophotometer.
`
`[0044]
`
`1020 ng of this size‘fractionated DNA was amplified by PCR with
`
`primers designed to amplify short STR regions (eg, D2281045, CSFlPO, D25441 see
`
`Table l for detail).
`
`{0045}
`
`Typical PCR reaction components were:
`
`10 mM dNTP
`
`25 mM MgCl2
`
`50 mM Primers
`
`Template 1 ugi’ul
`
`Ampli Taq Gold
`
`10X PCR Buffer
`
`2.0 pl
`
`1.5 pl
`
`0.5 ul
`
`2.0 ul
`
`0.5 111
`
`2.5 ul
`
`Water
`
`16.0 pl
`
`25
`
`30
`
`

`

`WO 2008/070862
`
`PCT/U52007/086862
`
`[00461
`
`Typical PCR cycle consisted of: Denaturation temperature of 94 EC for 30
`
`sec, annealing temperature varied from 56 to 62°C depending upon the primer length,
`
`extension was done at 72 DC. Number of cycles used ranged from 26 to 40‘
`
`[0047}
`
`These primers were also used for PCR reaction with the DNA extracted
`
`from fetal tissues and the total, unfractionated, mucous DNA. Shown in FIG. 2 and FIG. 3
`
`are PCR electropherograms that demonstrating that the 50-200 base pair DNA fraction
`
`resulted in the same fetal alleles as seen in fetal tissue PCR.
`
`Example 3
`
`[0048]
`
`This Example demonstrates that the mini-STR markers detect fetal alleles.
`
`10
`
`[0049]
`
`Mini-STR markers of the invention were used to detect fetal alleles from
`
`DNA extracted from clinical cervical mucous samples. Table 1, below, summarizes the
`
`results obtained. D151677-F and —R, D2281045-F and —R, D1081248—F and ~R, TPOX,
`
`Mini—LFG33—F and -R, and Mini-LFG34-F and 4R are exemplary primers of the invention.
`
`

`

`WO 2008/070862
`
`PCT/U52007/086862
`
`
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