Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

• the present disclosure relates to methods for analyzing fetal DNA. According to specific aspects, the disclosure relates to methods of isolating DNA from fetal extravillous trophoblast cells from a pregnant subject and analyzing the fetal DNA.
• fetal fraction refers to the fraction of DNA in a sample which is fetal DNA, wherein the remainder is maternal DNA.
• the fetal fraction relates conversely to the term "maternal contamination.”
• the sample is characterized by 90% maternal contamination.
• Endocervical samples obtained during an ongoing pregnancy are unique among fetal cell-containing samples.
• Extravillous trophoblast cells of fetal origin are naturally shed into the lower uterine pole. While it would seem that an endocervical sample containing these fetal extravillous trophoblast cells would be a useful source of fetal genomic DNA for analysis of the fetal genome, it has not been possible to reliably analyze fetal genomic DNA obtained by traditional DNA extraction from fetal extraviUous trophoblasts.
• sequencing of fetal DNA isolated from highly- purified fetal extraviUous trophoblast cells fails to produce a reliable fetal DNA signal.
• Endocervical samples obtained during an ongoing pregnancy contain many more maternal cells than fetal trophoblast cells.
• the inventors consider it possible that maternal DNA is released by senescent maternal cells in the endocervical samples and that this maternal DNA, likely degraded, is associated with the plasma membranes of fetal extraviUous trophoblast cells in the endocervical samples.
• Methods of isolating DNA of a fetus of an ongoing pregnancy include: obtaining a maternal endocervical sample containing maternal cells and fetal extraviUous trophoblast cells from a pregnant subject; isolating fetal extraviUous trophoblast cells from the maternal endocervical sample, producing isolated fetal extraviUous trophoblast cells contaminated with maternal DNA; lysing the isolated fetal extraviUous trophoblast cells; isolating fetal nuclei from the lysed fetal extraviUous trophoblast cells, producing isolated fetal nuclei, thereby removing at least a portion of the contaminating maternal DNA; lysing the isolated fetal nuclei; and purifying genomic DNA from the isolated fetal nuclei, producing purified fetal genomic DNA.
• Methods of isolating DNA of a fetus of an ongoing pregnancy include: obtaining a maternal endocervical sample containing maternal cells and fetal extraviUous trophoblast cells from a pregnant subject; isolating fetal extraviUous trophoblast cells from the maternal endocervical sample, producing isolated fetal extraviUous trophoblast cells contaminated with maternal DNA; lysing the isolated fetal extraviUous trophoblast cells; isolating fetal nuclei from the lysed fetal extraviUous trophoblast cells, producing isolated fetal nuclei, thereby removing at least a portion of the contaminating maternal DNA; further treating the isolated fetal nuclei to remove at least a further portion of the contaminating maternal DNA; lysing the isolated fetal nuclei; and purifying genomic DNA from the isolated fetal nuclei, producing purified fetal genomic DNA.
• Methods of isolating DNA of a fetus of an ongoing pregnancy include: obtaining a maternal endocervical sample containing maternal cells and fetal extravillous trophoblast cells from a pregnant subject; isolating fetal extravillous trophoblast cells from the maternal endocervical sample, producing isolated fetal extravillous trophoblast cells contaminated with maternal DNA; lysing the isolated fetal extravillous trophoblast cells; isolating fetal nuclei from the lysed fetal extravillous trophoblast cells, producing isolated fetal nuclei, thereby removing at least a portion of the contaminating maternal DNA; further treating the isolated fetal nuclei to remove at least a further portion of the contaminating maternal DNA; lysing the isolated fetal nuclei; and purifying genomic DNA from the isolated fetal nuclei, producing purified fetal genomic DNA.
• the isolated fetal nuclei are treated with a DNAse prior to lysing the isolated fetal nuclei, thereby removing at least a portion of the contaminating maternal DNA.
• the isolated extravillous trophoblast cells are treated with a DNAse prior to isolating fetal extravillous trophoblast cells from the maternal endocervical sample, thereby removing at least a portion of the contaminating maternal DNA.
• the isolated extravillous trophoblast cells are treated with a DNAse prior to lysing isolated fetal extravillous trophoblast cells, thereby removing at least a portion of the contaminating maternal DNA.
• the fetal extravillous trophoblast cells are fixed prior to treating the fetal extravillous trophoblast cells with the DNAse and the DNAse is attached to a support, preventing entry of the DNAse into the fetal extravillous trophoblast cells.
• the fetal extravillous trophoblast cells are not fixed prior to treating the fetal extravillous trophoblast cells with the DNAse.
• the purified fetal genomic DNA is characterized by a fetal fraction in the range of 10%- 100%.
• the purified fetal genomic DNA is characterized by a fetal fraction in the range of 25%- 100%.
• Methods of assaying DNA of a fetus of an ongoing pregnancy include: obtaining a maternal endocervical sample containing fetal extravillous trophoblast cells from a pregnant subject; isolating fetal extravillous trophoblast cells from the maternal endocervical sample; lysing the isolated fetal extravillous trophoblast cells; isolating fetal nuclei from the lysed fetal extravillous trophoblast cells; lysing the isolated fetal nuclei and purifying genomic DNA from the isolated fetal nuclei; and assaying the purified fetal genomic DNA, thereby determining a characteristic of a genomic DNA sequence of the purified fetal genomic DNA, and thereby assaying genomic DNA of a fetus of an ongoing pregnancy.
• assaying the purified fetal genomic DNA determines a characteristic of at least one individual nucleotide in a genomic DNA sequence of the purified fetal genomic DNA, thereby assaying DNA of a fetus of an ongoing pregnancy with single base resolution.
• assaying the purified fetal genomic DNA includes performing a method selected from the group consisting of: sequencing, high resolution melt analysis, methylation analysis, capillary electrophoresis, mass spectrometry, single strand conformation polymorphism, single base extension, restriction fragment length polymorphism using the purified fetal genomic DNA.
• sequencing the purified fetal genomic DNA includes performing a method selected from the group consisting of: massively parallel signature sequencing, single-molecule real-time sequencing, polony sequencing, ion semiconductor, pyrosequencing, sequencing by synthesis, sequencing by ligation and chain termination sequencing.
• methods of assaying DNA of a fetus of an ongoing pregnancy include: obtaining a maternal endocervical sample containing fetal extravillous trophoblast cells from a pregnant subject; isolating fetal extraviUous trophoblast cells from the maternal endocervical sample; treating the isolated fetal nuclei to remove at least a portion of the contaminating maternal DNA; lysing the isolated fetal extraviUous trophoblast cells; isolating fetal nuclei from the lysed fetal extraviUous trophoblast cells; lysing the isolated fetal nuclei and purifying genomic DNA from the isolated fetal nuclei; and assaying the purified fetal genomic DNA, thereby determining a characteristic of a genomic DNA sequence of the purified fetal genomic DNA, and thereby assaying genomic DNA of a fetus of an ongoing pregnancy.
• methods of assaying DNA of a fetus of an ongoing pregnancy include treating the isolated extraviUous trophoblast cells with a DNAse prior to isolating fetal extraviUous trophoblast cells from the maternal endocervical sample, thereby removing at least a portion of the contaminating maternal DNA.
• methods of assaying DNA of a fetus of an ongoing pregnancy include treating the isolated extraviUous trophoblast cells with a DNAse prior to lysing isolated fetal extraviUous trophoblast cells, thereby removing at least a portion of the contaminating maternal DNA.
• Methods of assaying DNA of a fetus of an ongoing pregnancy include: obtaining a maternal endocervical sample containing fetal extraviUous trophoblast cells from a pregnant subject; isolating fetal extraviUous trophoblast cells from the maternal endocervical sample; lysing the isolated fetal extraviUous trophoblast cells; isolating fetal nuclei from the lysed fetal extraviUous trophoblast cells; lysing the isolated fetal nuclei and purifying genomic DNA from the isolated fetal nuclei; and assaying the purified fetal genomic DNA, wherein the fetal extraviUous trophoblast cells are fixed prior to treating the fetal extraviUous trophoblast cells with the DNAse and the DNAse is attached to a support, preventing entry of the DNAse into the fetal extraviUous trophoblast cells, thereby determining
• Methods of assaying DNA of a fetus of an ongoing pregnancy are provided according to the present invention which include: obtaining a maternal endocervical sample containing fetal extraviUous trophoblast cells from a pregnant subject; isolating fetal extraviUous trophoblast cells from the maternal endocervical sample; lysing the isolated fetal extraviUous trophoblast cells; isolating fetal nuclei from the lysed fetal extraviUous trophoblast cells; lysing the isolated fetal nuclei and purifying genomic DNA from the isolated fetal nuclei; and assaying the purified fetal genomic DNA, wherein the fetal extraviUous trophoblast cells are not fixed prior to treating the fetal extraviUous trophoblast cells with the DNAse, thereby determining a characteristic of a genomic DNA sequence of the purified fetal genomic DNA, and thereby assaying genomic DNA of a fet
• the purified fetal genomic DNA is characterized by a fetal fraction in the range of 10%- 100%.
• the purified fetal genomic DNA is characterized by a fetal fraction in the range of 25%- 100%.
• Figure 1 is an image of a gel which shows amplified DNA of fetal and maternal cells in five independent samples, plus positive and negative controls; the amplified DNA is depicted as a smear between 700 and 900 base pair length, as shown by the DNA standard size markers (L);
• Figure 2 is an image of a gel which shows PCR amplified fragments from various chromosomes in fetal and maternal cells from five independent samples, as well as positive and negative controls; the lengths of the fragments are between 100-800 base pairs, as indicated by the DNA standards (L);
• Figure 3 is an image of a gel which shows digital electrophoretic analyses of successful library preparation from fetal (F) and maternal (M) cells in five independent samples; sizes of the DNA standard size markers (L) are indicated on the y-axis for reference;
• Figure 4 is an image of a Cercos heat map that indicates mean coverage by targeted regions ( ⁇ 1 Mb) of all exome sequences on log 10 scale, indicating each chromosome (1-22, X, Y) in maternal (M) and fetal (F) samples; the expanded region on the left shows detailed coverage for X and Y chromosomes;
• Figure 5A is a graph showing the relative coverage of autosomal, X and Y chromosomes after WGA for fetal and maternal cells
• Figure 5B is a graph showing the relative coverage of autosomal, X and Y chromosomes after WGA for newborn blood samples (bloodspots) and maternal blood samples which are used reference standards;
• Figure 6 is a graphical short tandem repeat (STR) profile of DNA obtained from maternal cells (Maternal), fetal trophoblast cells (TRIC), and placental tissue (Placenta) from one patient;
• STR short tandem repeat
• Figure 7 is a graph showing a comparison of allelic frequencies for the 94 sequenced SNPs in corresponding maternal and fetal (TRIC) specimens for a single patient;
• Figure 8 is a graph showing a comparison of allelic frequencies for the 94 sequenced SNPs in corresponding fetal (TRIC) and placental specimens for a single patient;
• Figure 9 is a graph showing a comparison of allelic frequencies for the 94 sequenced SNPs in corresponding maternal and placental specimens for a single patient.
• RNA Interference Nuts and Bolts of RNAi Technology, DNA Press LLC, Eagleville, PA, 2003; Herdewijn, P. (Ed.), Oligonucleotide Synthesis: Methods and Applications, Methods in Molecular Biology, Humana Press, 2004; A. Nagy, M. Gertsenstein, K. Vintersten, R. Behringer, Manipulating the Mouse Embryo: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press; December 15, 2002, ISBN- 10: 0879695919; Kursad Turksen (Ed.), Embryonic stem cells: methods and protocols in Methods Mol Biol.
• TRIC fetal trophoblasts obtained by a TRIC "Trophoblast Retrieval and Isolation form the Cervix" method.
• TRIC is a method of safe non-invasive fetal cell isolation from the cervical canal in ongoing pregnancies, see description in the examples.
• Methods for isolation of genomic DNA from cells of a fetus of an ongoing pregnancy and assay of the fetal genomic DNA are provided according to aspects of the present invention.
• Analysis of fetal cell genomic DNA provides detailed information about the fetus, such as detection of fetal abnormalities or variations, including single nucleotide polymorphisms, base modifications and detailed genomic DNA sequence information.
• Methods according to aspects of the present invention include purifying genomic DNA from isolated fetal extravillous trophoblast cell nuclei, producing purified fetal genomic DNA wherein the fetal fraction is in the range of 10% - 100%. Methods according to aspects of the present invention include purifying genomic DNA from isolated fetal extravillous trophoblast cell nuclei, producing purified fetal genomic DNA wherein the fetal fraction is in the range of 25% - 100%. Methods according to aspects of the present invention include purifying genomic DNA from isolated fetal extravillous trophoblast cell nuclei, producing purified fetal genomic DNA wherein the fetal fraction is 50% or greater.
• Methods according to aspects of the present invention include purifying genomic DNA from isolated fetal extravillous trophoblast cell nuclei, producing purified fetal genomic DNA wherein the fetal fraction is 75% or greater. Methods according to aspects of the present invention include purifying genomic DNA from the isolated fetal extravillous trophoblast cell nuclei, producing purified fetal genomic DNA wherein the fetal fraction is 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater.
• the purified fetal genomic DNA is characterized as having from 0% - 90% maternal contamination.
• Methods according to aspects of the present invention include purifying genomic DNA from the isolated fetal extravillous trophoblast cell nuclei, producing purified fetal genomic DNA wherein the maternal contamination is 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less.
• Methods according to aspects of the present invention include purifying genomic DNA from the isolated fetal extraviUous trophoblast cell nuclei, producing purified fetal genomic DNA wherein the maternal contamination is 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less.
• Methods of the present invention allow for purification and assay of fetal genomic DNA in spite of the overwhelming maternal cell population in an endocervical sample.
• Methods of the present invention provide, for the first time, clinically relevant fetal genomic data as early as gestational age four to five weeks from ongoing pregnancies. Gestational age is well-known to be defined as the time measured from the date of onset of the last menstrual period. Methods of the present invention are not hampered by factors such as body mass index or gestational age which complicate cell- free DNA assessment methods.
• compositions and methods described herein with particular reference to human females and human fetuses they are not limited to humans and fetal genomic DNA of other species may be similarly isolated and analyzed.
• Methods according to aspects of the present invention include obtaining a maternal endocervical sample containing fetal extraviUous trophoblast cells from a pregnant subject; isolating fetal extraviUous trophoblast cells from the maternal endocervical sample; lysing the isolated fetal extraviUous trophoblast cells; isolating fetal extraviUous trophoblast cell nuclei from the lysed fetal extraviUous trophoblast cells; and purifying genomic DNA from the isolated fetal extraviUous trophoblast cell nuclei, thereby isolating the fetal genomic DNA from extraviUous trophoblast cells.
• a maternal endocervical sample is collected from a pregnant female from gestational age of about 4 weeks to about 30 weeks of pregnancy, such as in the first trimester, second trimester and/or third trimester of pregnancy.
• a sample is collected from a pregnant subject at about two weeks after conception (gestational age 4 weeks) up to about 20 weeks of gestation (mid-point of pregnancy) or later.
• isolating fetal extravillous trophoblast cells from the maternal endocervical sample is accomplished by contacting the fetal extravillous trophoblast cells with an antibody specific for the fetal extravillous trophoblast cells, wherein the antibody does not bind to maternal cells in the maternal endocervical sample, and capturing the fetal extravillous trophoblast cells attached to the antibodies.
• the antibody is specific for major histocompatibility complex, class I, G (HLA-G).
• the antibody specific for the fetal extravillous trophoblast cells is attached to any solid or semi-solid support which is insoluble in aqueous solutions. Attachment of the antibody to the support is achieved by any of various methods, illustratively including adsorption to the support and chemical bonding to the support.
• the antibody specific for the fetal extravillous trophoblast cells can be directly or indirectly attached to a support.
• the term “directly attached” is used to indicate that the support is covalently or non-covalently bound to the antibody specific for the fetal extravillous trophoblast cells and that the support is not bound to the antibody via a secondary antibody.
• the term “indirectly attached” is used to indicate that the antibody specific for the fetal extravillous trophoblast cells is covalently or non- covalently bound to the support via an intermediate, such as a secondary antibody or linker.
• the antibody specific for the fetal extravillous trophoblast cells is indirectly bound to the support via binding of the antibody to a Protein A or Protein G molecule, wherein the Protein A or Protein G molecule is bound to the support.
• the insoluble solid or semi-solid support can be any of various materials such as glass; plastic, such as polypropylene, polystyrene, nylon; paper; silicon; nitrocellulose; or any other material to which a desired material can be attached without significant inhibition of the function of the material.
• the support can be in any of various forms or shapes, including planar, such as silicon chips and glass plates; and three-dimensional, such as particles, microtiter plates, microtiter wells, pins, fibers and the like.
• an insoluble solid or semi-solid support is a particle.
• the particles can be of any shape, such as cylindrical, spherical, and so forth, size, composition, or physiochemical characteristics.
• the particle size or composition can be chosen so that the particle can be separated from fluid, e.g., on a filter with a particular pore size or by some other physical property.
• the particles used can have a diameter of less than one millimeter, for example, a size ranging from about 0.1 to about 1,000 micrometers in diameter, inclusive, such as about 3-25 microns in diameter, inclusive, or about 5-10 microns in diameter, inclusive, 1 nanometer (nm) to about 100,000 nm in diameter, inclusive, for example, a size ranging from about 10-1,000 nm, inclusive, or for example, a size ranging from 200-500 nm, inclusive.
• particles used are beads, particularly microbeads and nanobeads.
• An insoluble solid or semi-solid support can include functional groups for binding to a material to be bound to the support.
• a support can include carboxyl, amine, amino, carboxylate, halide, ester, alcohol, carbamide, aldehyde, chloromethyl, sulfur oxide, nitrogen oxide, epoxy and/or tosyl functional groups.
• Functional groups, modification thereof and binding of a material, such as an antibody or enzyme, to a support are known in the art.
• l-ethyl-3-[3- dimethylaminopropyl]carbodiimide hydrochloride, EDC or EDAC chemistry can be used to attach a desired material to an insoluble solid or semi-solid support.
• an antibody specific for the fetal extravillous trophoblast cells is directly attached to a plurality of magnetic particles and removing fetal extravillous trophoblast cells from the maternal endocervical sample includes exposure of the magnetic particles to a magnet.
• Magnetic nanoparticles directly coupled to the antibody which specifically binds to a fetal antigen typically have a size in the range of 10 nm - 1 um, although smaller or larger magnetic nanoparticles may be used.
• HLA-G antibody is attached to magnetic nanoparticles.
• cells of the maternal endocervical sample are fixed by treatment with a fixative, wherein the treatment with the fixative is performed prior to or following isolating fetal extravillous trophoblast cells from the maternal endocervical sample.
• the fixative used can be glutaraldehyde; formaldehyde; paraformaldehyde; or a combination of any two or more thereof.
• the aldehyde fixative is paraformaldehyde.
• the fixative is a non-aldehyde fixative.
• Non-aldehyde fixatives illustratively include acetone, acetic acid, and alcohols such as ethanol and methanol. Combinations of two or more non-aldehyde fixatives are optionally used. According to aspects of the present invention, a mixture of methanol and acetic acid is used as a non-aldehyde fixative.
• the maternal endocervical sample is first fixed in a non-aldehyde fixative.
• the isolated fetal extravillous trophoblast cells are then optionally fixed with an aldehyde fixative.
• the non-aldehyde fixative and/or aldehyde fixative is optionally removed or partly removed by washing the maternal endocervical sample with a physiological liquid or buffer, such as saline or a buffer compatible with mammalian cells.
• the fetal extravillous trophoblast cells are treated with a protease and/or a glycosaminoglycan degrading enzyme (GAGase), wherein treating the fetal extravillous trophoblast cells with a protease and/or a GAGase is performed prior to or following removing fetal extravillous trophoblast cells from the maternal endocervical sample and prior to or following treatment of the fetal extravillous trophoblast cells with a nuclease.
• GAGase glycosaminoglycan degrading enzyme
• Glycosaminoglycan degrading enzymes include, for example, hyaluronidase, heparinase and chondroitinase.
• the maternal endocervical sample is not treated with a mucolytic agent.
• the maternal endocervical sample is not treated with a mucolytic agent selected from N-acetyl-L-cysteine, DTT, trypsin and trypsin/EDTA prior to isolation of the fetal extravillous trophoblasts.
• the maternal endocervical sample is not treated with one or more of a collagenase, a protease, a liberase blendzyme, and a mucolytic agent prior to isolation of the fetal extravillous trophoblasts.
• a maternal endocervical sample is acidified prior to isolating fetal extravillous trophoblast cells.
• An acidifying agent is optionally added to the sample bringing the pH of the sample to about pH 5 - 6.
• An acidifying agent can be any acid or acidic buffer, for example.
• the isolated fetal extravillous trophoblast cells are further processed to isolate nuclei from the fetal extravillous trophoblast cells.
• the isolated fetal extravillous trophoblast cells are lysed to break open the cells and release intact nuclei by any of various methods including, but not limited to, a physical, chemical, microfluidic or mixed modality method of breaking open the cells and releasing intact nuclei.
• Physical cell lysis methods include, but are not limited to, homogenization, sonication, vortexing and beadbeating. Homogenization methods for lysis of cells and release of intact nuclei are exemplified by Dounce homogenization.
• Non-ionic detergents used to lyse cells and release intact nuclei include, but are not limited to, nonyl phenoxypolyethoxylethanol (NP-40), typically in the range of 0.1 - 1%, octylphenoxypolyethoxyethanol and derivatives such as branched octylphenoxy poly(ethyleneoxy)ethanol (IGEPAL CA-630), typically in the range of 0.1
• Triton X-100 t-octylphenoxypolyethoxyethanol
• the non-ionic detergent is present in a hypotonic buffer.
• An example cell lysis buffer to lyse isolated fetal extravillous trophoblast cells and release intact nuclei is 10 mM Hepes pH 7.5, 2 mM MgCl 2 , 25 mM KC1, 0.5% NP-40 or 1% Triton X- 100.
• Chemical cell lysis methods include protease treatment, such as treatment with a protease including, but not limited to, protease K, pronase, trypsin and/or pepsin.
• protease treatment such as treatment with a protease including, but not limited to, protease K, pronase, trypsin and/or pepsin.
• the activity of a protease depends on concentration of the protease, pH, incubation time and temperature, all of which can be adjusted by the skilled artisan to achieve the desired extent of proteolysis.
• enzymes with limited protein cleavage sites such as pepsin (PhelVal, Gln4His, Glul3Ala, Alal4Leu, Leul5Tyr, Tyrl6Leu, Gly23Phe, Phe24) or trypsin are used to gradually digest the cell membrane without affecting the nucleus such that the nuclear DNA remains intact.
• cells are subjected to homogenization in a hypotonic buffer with a non-ionic detergent to lyse the cells and release intact nuclei.
• Microfluidic methods can be used to lyse cells and release intact nuclei, for example as described in U.S. Patent No. 8304185; Nan et al., Lab Chip, 14: 1060-1073, 2014; and Cui et al., Ann. Rev. Biomed. Engin., 17:267-286, 2015.
• Cell lysate containing intact nuclei is further processed to isolate the intact nuclei. For example, separation of fetal extravillous trophoblast cell nuclei is accomplished by dissection, washing, differential centrifugation, filtration or microfluidic separation of the intact nuclei from other components present in the cell lysate.
• Isolation of fetal extravillous trophoblast cell nuclei optionally includes centrifugation to pellet the nuclei and removal of the supernatant containing fetal extravillous trophoblast cell cytoplasm.
• the nuclei can be washed and centrifugation repeated one or more times.
• Microfluidic techniques can be used to separate fetal extravillous trophoblast cell nuclei from other components present in the cell lysate, for example as described in U.S. Patent No. 8304185; Nan et al., Lab Chip, 14: 1060-1073, 2014; and Cui et al., Ann. Rev. Biomed. Engin., 17:267-286, 2015.
• fetal extravillous trophoblast cells are affixed to an insoluble solid or semi-solid support, the cells lysed and the cytoplasm washed away, leaving the nuclei on the support.
• Any support can be used, such as those mentioned herein.
• a glass slide is a specific example of a support that can be used. Cells are easily attached to glass slides by centrifugation of the slide in contact with a sample containing cells.
• a drop of liquid containing cells can be placed on a slide.
• the cells attach to the glass slide without further treatment.
• a solid or semi-solid support is optionally treated with an adherence promoter to promote adherence of cells.
• Adherence promoters are exemplified by polycationic materials such as poly-L-lysine and poly-L-inosine; and/or extracellular matrix materials exemplified by collagen, fibronectin and laminin.
• an antibody specific for a nuclear protein of an intact nucleus accessible to the antibody without lysis of the nuclei is bound to the nuclei in order to separate the nuclei.
• the antibody can be bound to a support, such as those supports described herein, such that nuclei bound to the antibody are indirectly bound to the support and other materials can then be washed away.
• the antibody can be attached to a magnetic support, such as magnetic particles, allowing a nucleus bound to the antibody attached to a magnetic support to be separated from other components by exposure to a magnet.
• Antibodies specific for a nuclear protein of an intact nucleus accessible to the antibody without lysis of the nucleus include, but are not limited to, antibodies specific for nesprin proteins which are located on the surface of the outer nuclear membrane.
• nuclei are isolated from the fetal extravillous trophoblast cell lysate by size exclusion methods, such as filtration, which allows retention of nuclei and removal of other lystate components based on difference in size.
• the isolated fetal extravillous trophoblast cell nuclei are lysed by any of various methods including, but not limited to, a physical, chemical, microfluidic or mixed modality method of breaking open the nuclei and releasing genomic DNA.
• Physical methods for lysis of nuclei include, but are not limited to, homogenization, sonication, vortexing and beadbeating. Homogenization methods for lysis of nuclei and release of genomic DNA are exemplified by Dounce homogenization.
• Chemical cell lysis methods for lysis of nuclei and release of genomic DNA include, but are not limited to, treatment of cells with one or more detergents and/or chaotropic agents.
• Lysis of isolated nuclei optionally includes treatment with a hypotonic, hypertonic buffer and/or an ionic detergent, such as SDS, typically in the range of 0.1 - 1%.
• the ionic detergent is present in a hypotonic buffer.
• An example lysis buffer to lyse isolated nuclei is 400 mM NaCl, 10 mM Tris, 2 mM EDTA, pH 8.2, 0.66% SDS and 50 ⁇ g/ml proteinase K.
• Isolated nuclei may be subjected to homogenization in a hypotonic buffer with an ionic detergent to lyse the nuclei.
• Chemical nuclei lysis methods include protease treatment, such as treatment with a protease including, but not limited to, protease K, pronase, trypsin and/or pepsin.
• protease treatment such as treatment with a protease including, but not limited to, protease K, pronase, trypsin and/or pepsin.
• the activity of a protease depends on concentration of the protease, pH, incubation time and temperature, all of which can be adjusted by the skilled artisan to achieve the desired extent of proteolysis.
• nuclei are affixed to an insoluble solid or semi-solid support, the nuclei lysed and the nuclear contents washed away, leaving the genomic DNA on the support.
• Any support can be used, such as those mentioned herein.
• a glass slide is a specific example of a support that can be used.
• the fetal genomic DNA is isolated from the lysate of the nuclei by DNA isolation methods, exemplified by precipitation, centrifugation and washing; electrophoretic isolation such as gel electrophoresis; size exclusion chromatography; gradient centrifugation such as step gradient centrifugation or continuous gradient centrifugation.
• the maternal endocervical samples, isolated fetal extravillous trophoblast cells and/or isolated fetal extravillous trophoblast cell nuclei are optionally treated to remove at least a portion of maternal DNA present in the sample but not contained in the maternal cells.
• Treatments to remove maternal DNA include, but are not limited to, treatment with a DNAse to digest the maternal DNA. The digested maternal DNA can then be washed away, thereby removing at least a portion of the maternal DNA.
• the maternal DNA is contacted with a DNA absorbant material to absorb the maternal DNA. The absorbant material with absorbed maternal DNA is then removed, thereby removing at least a portion of the maternal DNA.
• a DNA absorbant material is exemplified by anti-DNA antibodies, optionally attached to a support.
• the maternal endocervical sample including maternal cells and fetal extravillous trophoblast cells is optionally treated with a DNAse and/or protease, producing a DNAse-treated sample and/or a protease-treated sample. If the cells are fixed prior to DNAse and/or protease treatment, at least the DNAse, and optionally the protease, is attached to a support which cannot enter the fixed cells.
• active DNase and/or protease is removed from the DNAse-treated sample and/or a protease-treated sample, such as by filtration, washing and/or heat-inactivation of the DNase and/or protease.
• the isolated fetal extravillous trophoblast cells Prior to lysis, are optionally treated with a DNase and/or protease. If the cells are fixed prior to DNAse and/or protease treatment, at least the DNAse, and optionally the protease, is attached to a support which cannot enter the fixed cells. Following treatment of the fetal extravillous trophoblast cells with a DNase and/or protease, active DNase and/or protease is removed from the DNAse-treated cells and/or a protease-treated cells, such as by filtration, washing and/or heat-inactivation of the DNase and/or protease.
• intact fetal extraviUous trophoblast cell nuclei released from the cells are optionally treated with a DNAse and/or protease. If the nuclei are fixed prior to DNAse and/or protease treatment, at least the DNAse, and optionally the protease, is attached to a support which cannot enter the fixed nuclei.
• active DNase and/or protease is removed from the DNAse-treated nuclei and/or a protease-treated nuclei, such as by filtration, washing and/or heat-inactivation of the DNase and/or protease.
• DNAse inhibitors is optionally included in a wash buffer following treatment of cells and/or nuclei with DNAse and during removal of the DNAse from contact with the cells and/or nuclei to protect the nuclei and DNA.
• DNAse inhibitors are exemplified by 2-mercaptoethanol, 2-nitro-5-thiocyanobenzoic acid, actin, alfa B2a, G2, G2a and Ml (non-competitive); Ca2 + , EGTA, EDTA, sodium dodecyl sulfate (SDS), calf spleen inhibitor protein, carbodiimide, cholesterol sulfate, iodoacetate and combinations of any two or more thereof.
• a DNAse used in a method according to aspects of the present invention is optionally attached, such as by covalent linkage, to one or more supports, such as supports described herein, so that the DNAse cannot penetrate fixed cells or fixed nuclei.
• a DNAse is attached to particles, such as beads, sized to prevent entry into fixed cells. Such particles typically have a particle diameter great than lOnm.
• intact fetal extraviUous trophoblast cells are not treated with a DNAse before or after isolation from the maternal endocervical sample.
• isolated fetal extraviUous trophoblast nuclei are not treated with a DNAse before or after isolation from fetal extraviUous trophoblast cells.
• the fetal extraviUous trophoblast cell genomic DNA is assayed to determine one or more characteristics of the fetal extraviUous trophoblast cell DNA and is optionally compared to a standard. Assaying the fetal extraviUous trophoblast cell genomic DNA includes any applicable genomic DNA assay.
• Genomic DNA assays include, but are not limited to, sequencing, high resolution melt analysis, methylation analysis, capillary electrophoresis, mass spectrometry, single strand conformation polymorphism, single base extension and restriction fragment length polymorphism. Genomic DNA assays include assays to detect single nucleotide polymorphisms and assays of DNA base modifications with single base resolution such as detection of methylation at one or more single bases in a genomic DNA sequence.
• Sequencing methodologies include massively parallel sequencing, single- molecule real-time sequencing, polony sequencing, ion semiconductor (Ion Torrent sequencing), pyrosequencing (454), sequencing by synthesis (Illumina), sequencing by ligation (SOLiD sequencing), chain termination (Sanger sequencing), for example.
• Genomic DNA assays include, but are not limited to, dot blot; Southern blot; and DNase protection. Details of such assays are described in J. Sambrook and D.W. Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rd Ed., 2001; and F.M. Ausubel, Ed., Short Protocols in Molecular Biology, Current Protocols; 5th Ed., 2002, for example.
• Fetal genomic DNA is optionally amplified by an amplification method prior to performing a genomic DNA assay.
• Amplification methods include template directed primer extension mediated by a nucleic acid polymerase and a pair of primers which flank the target nucleic acid to be amplified, including, but not limited to, polymerase chain reaction (PCR), ligation-mediated PCR( LM-PCR), phi-29 PCR, real-time quantitative PCR (qPCR), whole genome amplification and other nucleic acid amplification methods, for instance, as described in C.W. Dieffenbach et al., PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2003; V.
• PCR polymerase chain reaction
• LM-PCR ligation-mediated PCR
• qPCR real-time quantitative PCR
• Methods according to aspects of the present invention include determining a characteristic of at least one individual nucleotide in a genomic DNA sequence of the purified fetal genomic DNA, thereby assaying DNA of a fetus of an ongoing pregnancy.
• Genomic DNA isolated from fetal cells is analyzed to identify genomic DNA variants, compared to a standard, in the fetal cells according to aspects of the present invention.
• Standards suitable for genomic DNA assays are well-known in the art and the standard used can be any appropriate standard.
• a standard may be a reference genome concurrently analyzed or previously determined in a sample of an individual control subject or in a population of control subjects and stored in a print or electronic medium for recall and comparison to a result of an assay.
• inventive compositions and methods are illustrated in the following examples. These examples are provided for illustrative purposes and are not considered limitations on the scope of inventive compositions and methods.
• Maternal and fetal cells were obtained from five cervical specimens at 5, 7, 14, 15 and 15 weeks of gestation. Maternal blood as well as fetal blood spot DNA from the same patients was used as reference material as available. Genomic DNA from five fetal/maternal pairs was isolated and exome sequencing performed.
• Endocervical cells were obtained with a ThinPrep and immediately fixed during the PAP smear procedure, preserving proteins and nucleic acids. The collected sample was processed to separate the mucus and cells. The cells were then washed with phosphate buffered saline (PBS), centrifuged and re suspended in 1.5 mL PBS, combined with anti-HLA-G antibody coated magnetic nanoparticles, and incubated overnight at 4°C with mixing. The HLA-G negative non-bound cells were collected after magnetic immobilization and three washings in PBS. The HLA-G positive bound cells are collected and stained for the EVT markers.
• PBS phosphate buffered saline
• the isolated HLA-G positive extravillous trophoblast (EVT) cells are referred to interchangeably as isolated fetal cells, isolated fetal extravillous trophoblast cells or isolated EVT herein and the HLA-G negative cervical cells as maternal cells.
• Isolated fetal extravillous trophoblast cells (HLA-G positive) were spun onto slides and stained for X and Y chromosome by the FISH technique. The samples that were positive for Y chromosome were selected for following experiments. The samples used are listed in Table I. Table I: The sample IDs identified as males based on FISH
• the fetal cell DNA was extracted from the slides used for FISH and the maternal cell DNA from the HLA-G negative cell fraction.
• Isolated fetal extravillous trophoblasts on slides that were not previously used for FISH analysis were treated as follows to remove cell membranes and cytoplasm potentially contaminated with maternal DNA leaving only isolated fetal extravillous trophoblast nuclei.
• Isolated fetal extravillous trophoblast cells on the slides were incubated in freshly prepared protease solution (0.011 gm pepsin in 100 mL 0.01N HC1) for 11 min at 37°C to lyse the cells while fetal nuclei remain intact. Cell debris was removed following lysis by washing in IX Phosphate buffered saline (PBS, pH 7.5), leaving the intact fetal nuclei on the slides.
• protease solution 0.011 gm pepsin in 100 mL 0.01N HC1
• the isolated fetal nuclei were then lysed by incubation with a PCR compatible lysis buffer, 5mM Tris ⁇ HCL, pH 8.8 including proteinase K, overnight at 42°C, followed by inactivation of the enzyme at 65°C for 30 mins and 80°C for 15 mins (The cell to buffer ratio was maintained at ⁇ buffer: 2 cells).
• a standard TaqMan real-time PCR (qPCR) assay with the Copy No. probe (Applied Biosystems) to validate the DNA extraction procedure and SRY probe (Applied Biosystems) to confirm the male gender of the isolated EVT (fetal samples).
• DNA from HLA-G negative maternal cells was extracted using a standard silica column based DNA purification protocol, commercially available as DNeasy Blood & Tissue Kit (Qiagen). DNA extracted was quantified using a fluorescent DNA intercalating dye assay, Pico Green Assay (Invitrogen).
• DNA extraction & purification from maternal and fetal blood samples was extracted using a standard silica column based DNA purification protocol, commercially available as DNeasy Blood & Tissue Kit (Qiagen). DNA extracted was quantified using a fluorescent DNA intercalating dye assay, Pico Green Assay (Invitrogen).
• Figure 1 shows amplified DNA of fetal and maternal cells from five independent samples, plus positive (+) and negative (-) controls.
• the amplified DNA is depicted as a smear between 700 and 900 base pairs in length, as shown by the DNA standard size markers (L).
• Figure 2 shows PCR amplified fragments from various chromosomes of fetal and maternal cells from five independent samples, plus positive (+) and negative (-) controls. The lengths of the fragments are between 100 - 800 base pairs in length, as shown by the DNA standard size markers (L).
• L DNA standard size markers
• L Ladder F: Fetal sample
• M Maternal sample.
• Controls, - Water control for amplification procedure
• + Amplified control human male genomic DNA
• Figure 2 shows a representative image.
• DNA libraries were prepared as per manufacturer's protocol using the Nextera Rapid Capture Exome Kit (Illumina) using 50ng of starting input of DNA.
• the samples and their respective fractions used for Library preparation are outlined in Table III.
• Table III Sample IDs and their representative fractions used for library preparation.
• Isolated fetal extravillous trophoblast cells were spun onto slides.
• the slides are viewed under bright field microscope to outline the area with cells.
• the slides are immersed in freshly prepared protease solution (0.011 gm pepsin in 100 niL 0.01N HC1) for l lmin at 37 C to lyse the cells and release intact fetal extravillous trophoblast nuclei.
• the slides are then washed IX Phosphate buffered saline (PBS, pH 7.5) for 5 min at room temperature.
• the slides are then dehydrated by passing them through alcohol gradients ethanol 90%, 80% and 70% each for lmin.
• the slides are then allowed to dry and viewed under the microscope again to ensure that there is no cell loss.
• nuclei lysis buffer in the tube is adjusted to maintain a ratio of 2cells/ ⁇ L of buffer.
• the slides are then visualized again to ensure all cells have been extracted.
• the tubes are then incubated overnight at 65°C followed by inactivation at 95°C for 30 mins.
• the success of DNA isolation is confirmed by TaqMan real-time PCR (qPCR) assay with the Copy No. probe (Applied Biosystems) and SRY probe (Applied Biosystems) to confirm the gender.
• the fetal extravillous trophoblast cell DNA was extracted from isolated nuclei on slides following cell lysis.
• Maternal cell DNA was isolated from the HLA-G negative cell fraction using 5 - lOul of 5 mM Tris/HCL pH 8.8/proteinase K cell lysis buffer overnight at 42°C and then inactivation at 65°C for 30 mins and 80°C for 15 mins.
• the cell to buffer ratio was maintained at ⁇ buffer: 2 cells.
• the number of cells lysed per sample is indicated in Table IV.
• the unstained slides are also viewed under bright field microscope to mark the area with cells.
• the slides are then immersed in freshly prepared protease solution (0.011 gm Pepsin in 0.01N HC1) for l lmin at 37°C.
• the slides are then washed IX PBS (Phosphate buffered saline, pH 7.5) for 5 min at room temperature.
• the slides are then dehydrated by passing them through alcohol gradients ethanol 90%, 80% and 70% each for lmin and allowed to dry.
• the slides are again viewed under the microscope again to ensure that there is no cell loss.
• the cells are lysed by adding 5ul of lysis buffer (Arcturus PicoPure DNA extraction kit, ThermoFischer) heated to 65 °C and the slide is placed on the thermocycler set at 65°C for 1-2 min. An additional 5 ⁇ 1 of lysis buffer is added and the total amount is removed from the slide and placed in a 0.2ml tube. The final amount of lysis buffer in the tube is adjusted to maintain a ratio of 2cells/luL of buffer. The slide is visualized to ensure that all the cells have been extracted off the slide. The tubes are then incubated overnight at 65°C followed by inactivation at 95°C for 30 mins. The success of DNA isolation is confirmed by TaqMan real-time PCR (qPCR) assay with the Copy No. probe (Applied Biosystems) and SRY probe (Applied Biosystems) to confirm the gender.
• lysis buffer Analogenzymerase
• Example 3 Isolation of genomic DNA from fetal extravillous trophoblast cells
• a maternal endocervical sample is collected using a cytobrush and the cytobrush is rinsed in ice-cold culture medium or PBS (137 mM NaCl, lOmM Phosphate buffer).
• Cells are centrifuged and resuspended in 10 ml PBS plus 2.7mM CaCl 2 , 1 mM MgCl 2 and then warmed to room temperature.
• the magnetic separation procedure for removing fetal cells is started by addition of 20 ul of 250 nm magnetic nanoparticles conjugated to an anti-HLA-G antibody to the washed cells after resuspension in 1 ml PBS and incubated at 4°C for 1 to 24 hours with shaking.
• Maternal cells (HLA-G negative) are separated from magnetized (HLA-G positive) fetal extravillous trophoblast cells using a DynaMagTM Spin magnet (Life Technologies).
• the fetal extravillous trophoblast cells are then washed 3 times using a magnet to remove residual maternal cells.
• the isolated fetal extravillous trophoblast cells are resuspended in a solution of 50-100 microliters PBS/10 mM EDTA. An aliquot of 15 microliters is removed for cell counting and quality control for fetal cells.
• the isolated maternal cells are resuspended in a solution of 50-100 microliters PBS/10 mM EDTA and an aliquot of 15 microliters is removed for cell counting and quality control, if desired.
• the isolated fetal extravillous trophoblast cells are lysed by addition of 50 mM Tris-HCl, pH 8.5, 150 mM NaCl, 1% NP-40, followed by centrifugation at low speed, about 1000 x g, to pellet intact fetal extravillous trophoblast cell nuclei. The supernatant is removed and the nuclei are washed with PBS/ 10 mM EDTA two or three times.
• Genomic DNA is extracted from the fetal extravillous trophoblast cell nuclei by addition of 25 microliters of 3X concentrated DNA extraction buffer (90 mM TRIS, 90 mM EDTA, 1.5% EDTA, pH 8.0, 3 mg/ml Proteinase K) to 50 microliters of the isolated fetal extravillous trophoblast cell nuclei. The nuclei are then incubated for 3 hrs at 65°C, followed by 10 min at 95°C.
• 3X concentrated DNA extraction buffer 90 mM TRIS, 90 mM EDTA, 1.5% EDTA, pH 8.0, 3 mg/ml Proteinase K
• the extracted genomic DNA is frozen for subsequent assays or can be further purified by centrifugation at high speed in a microcentrifuge for 5 minutes.
• the DNA is still further purified, for example using commercial DNA purification and concentration kits, such as a Zymresearch DNA 10 concentration kit.
• a maternal endocervical sample is collected using a cytobrush by inserting it approximately 2 cm into the endocervical canal and rotating 2 or 3 times as it is withdrawn. Mucus present in the canal is also collected in the brush.
• the cytobrush is rinsed in fixative, for example, using a ThinPrep kit.
• the specimen is stored refrigerated or at ambient temperature and transported to the laboratory for further processing. It can be stored at 4°C for at least one week without loss of RNA or HLA-G protein.
• the cells are centrifuged through a 250 micron filter and resuspended in 10 ml PBS at 4°C. The cells are then washed 2 more times in 10 ml PBS and the final pellet is brought to a volume of 1 ml in PBS.
• the magnetic separation procedure for removing fetal cells is started by addition of 20 ul of 250 nm magnetic nanoparticles conjugated to an anti-HLA-G antibody to the washed cells after resuspension in 1 ml PBS and incubated at 4°C for 1 to 24 hours with shaking.
• Maternal cells (HLA-G negative) are separated from magnetized (HLA-G positive) fetal extravillous trophoblast cells using a DynaMagTM Spin magnet (Life Technologies).
• the isolated fetal extravillous trophoblast cells are resuspended in a solution of 50-100 microliters PBS/10 mM EDTA. An aliquot of 15 microliters is removed for cell counting and quality control for fetal cells.
• the isolated maternal cells are resuspended in a solution of 50-100 microliters PBS/10 mM EDTA and an aliquot of 15 microliters is removed for cell counting and quality control, if desired.
• DNase I is prepared by dissolving 1 mg DNase I powder (Worthington Cat #2138, >2000 Kunitz units/mg) in 10 ml PBS plus 0.9 mM MgCl 2 .
• the DNAse is attached, such as by covalent linkage, to one or more supports such as beads or particles that cannot penetrate the fixed cells.
• DNAse covalently bound to non-magnetic beads is added to the isolated fetal extravillous trophoblast cells, 5 microliters of F7 (MoBiTec, Germany), and incubated for 10 mins at room temperature.
• the DNAse-treated isolated fetal extravillous trophoblast cells are centrifuged through a 250 micron filter and resuspended in 10 ml PBS at 4°C. The cells are then washed 2 more times in 10 ml PBS and the final pellet is brought to a volume of 1 ml in PBS. DNase immobilized on beads will be removed before fetal DNA extraction, using size exclusion methods such as gel filtration, filtration over meshes, laminar flow in microfluidic channels or other appropriate methods.
• the DNAse-treated isolated fetal extravillous trophoblast cells are lysed by addition of 50 mM Tris-HCl, pH 8.5, 150 mM NaCl, 1% NP-40, followed by centrifugation at low speed to pellet intact fetal extravillous trophoblast cell nuclei. The supernatant is removed and the nuclei are washed with PBS/ 10 mM EDTA two or three times.
• Genomic DNA is extracted from the fetal extravillous trophoblast cell nuclei by addition of 25 microliters of 3X concentrated DNA extraction buffer (90 mM TRIS, 90 mM EDTA, 1.5% EDTA, pH 8.0, 3 mg/ml Proteinase K) to 50 microliters of the isolated fetal extravillous trophoblast cell nuclei. The nuclei are then incubated for 3 hrs at 65 °C, followed by 10 min at 95 °C.
• 3X concentrated DNA extraction buffer 90 mM TRIS, 90 mM EDTA, 1.5% EDTA, pH 8.0, 3 mg/ml Proteinase K
• the extracted genomic DNA is frozen for subsequent assays or can be for further purified by centrifugation at high speed in a microcentrifuge for 5 minutes.
• the DNA is still further purified, for example using commercial DNA purification and concentration kits, such as a Zymresearch DNA 10 concentration kit.
• Endocervical samples were immediately fixed using a ThinPrep kit (Hologic, Marlborough, MA). The endocervical samples were centrifuged, re-suspended in 10 mL phosphate buffered saline (PBS), and then washed three times with PBS. After a final resuspension in 1.5 mL PBS, anti-HLA-G-coated magnetic nanoparticles were added and incubated overnight at 4°C with mixing. The non-bound (maternal) cells were collected after magnetic immobilization and separation of HLA-G-positive (fetal) cells. Isolated cells of both types were then washed three times in PBS.
• PBS phosphate buffered saline
• Trophoblast cells 110- 1515, average 373 from each sample
• maternal cells were counted, and groups of approximately 50 cells were mounted onto glass microscope slides.
• the cells were assessed by immunofluorescence microscopy for expression of ⁇ -hCG, and the percentage of labeled cells was determined.
• Expression of the trophoblast-specific protein, ⁇ -hCG was present in the isolated fetal cells in an average of 89.8 + 5.2% of the isolated fetal cells, while ⁇ -hCG was absent from isolated maternal cells.
• Table V shows examples from three of the samples that were assessed.
• FISH Fluorescence in situ hybridization
• Isolated fetal extravillous trophoblast cells were mounted on slides and probed for X and Y chromosomes by FISH using the DYZ1 satellite III on the Y chromosome, and the DXZ1 alpha satellite on the X chromosome as fluorescently- labeled probes (Abbott Molecular). Nuclei were counters tained with DAPI and scored for each chromosome to quantify cells that were XX or XY. Fluorescence in situ hybridization (FISH) for the X and Y chromosomes and quantitative PCR (qPCR) for the SRY gene identified trophoblast isolates from male fetuses.
• FISH Fluorescence in situ hybridization
• qPCR quantitative PCR
• Fetal DNA was obtained by isolation of fetal extravillous trophoblast nuclei prior to extracting fetal DNA from the nuclei. Isolated fetal extravillous trophoblast cells were mounted on slides, lysed, and fetal cell DNA was extracted from nuclei which remained attached to the slides.
• the cells were lysed to release intact nuclei by incubation in freshly prepared protease solution (0.011 gm pepsin in 100 mL 0.01N HC1) for 11 min. at 37°C to lyse the cells and release intact fetal extravillous trophoblast nuclei.
• protease solution 0.011 gm pepsin in 100 mL 0.01N HC1
• nuclei were lysed by incubation in 5 - lOul of 5 mM Tris/HCL pH 8.8/proteinase K cell lysis buffer overnight at 42°C and the cell to buffer ratio was maintained at lpL buffer: 2 cells. After inactivation at 65°C for 30 mins and 80°C for 15 mins, 1 iL was used for qPCR of RNaseH and SRY genes to confirm DNA extraction from the nuclei and the male gender of the fetal cells.
• the corresponding maternal cell DNA was extracted from 20-cell aliquots of maternal cells from the corresponding endocervical samples, depleted of fetal cells by immunomagnetic separation, that had been stored frozen in PCR tubes.
• Targeted sequencing was performed on DNA isolated from cells treated with DNAse. Fetal and maternal cells isolated from endocervical samples were dropped on separate slides and allowed to dry, adhering them to the slides.
• the slides with adhered fetal cells were immersed in freshly prepared protease solution (0.011 gm pepsin in 100 mL 0.01N HC1) for 11 min at 37°C to lyse the cells and release intact fetal extravillous trophoblast nuclei, followed by a PBS wash for 5 mins to remove cell plasma membranes and potential maternal DNA fragments, producing isolated fetal extravillous trophoblast nuclei.
• protease solution 0.011 gm pepsin in 100 mL 0.01N HC1
• non-fetal DNA was further eliminated from the glass-bound isolated fetal extravillous trophoblast nuclei by adding 10 ⁇ ⁇ of washed, immobilized DNase (DNase I, immobilized on matrix F7M, MoBiTec, Goettingen, Germany) onto the pepsin-treated slides, and incubating for 3-5 mins at room temperature. The slides were washed with PBS to terminate DNAse activity by removing the DNAse-containing beads.
• DNase I immobilized on matrix F7M, MoBiTec, Goettingen, Germany
• the isolated fetal extravillous trophoblast nuclei were then lysed by incubation overnight at 65°C with 0.5 ⁇ / ⁇ 5 mM Tris/HCL pH 8.8/proteinase K nuclei lysis buffer overnight at 42°C and the cell to buffer ratio was maintained at ⁇ , buffer: 2 cells. The protease was then inactivated by incubation at 95°C for 30 min.
• Placental villi (20-25 mg) were dissected and suspended in 100 ⁇ , PBS. Ten ⁇ L ⁇ of immobilized DNase was added and shaken for 10 min at room temperature to ensure efficient DNA digestion. DNase beads were removed by washing in PBS. DNA was extracted from the tissue, using a standard DNA isolation method.
• DNA from 10,000 maternal cells isolated from the endocervical samples was extracted, using a standard silica column based DNA purification protocol, commercially available as DNeasy Blood & Tissue Kit (Qiagen). All isolated DNA was quantified using a fluorescent DNA intercalating dye assay, Pico Green Assay (Invitrogen).
• Fetal genomic DNA was isolated from 10-50 nuclei, see Table V, and aliquots of fetal or maternal DNA ( ⁇ 4 genome equivalents) were used for whole genome amplification (WGA). WGA was confirmed by PCR amplification of four autosomal chromosome fragments, see Figures 1 and 2. A portion of DNA (approximately 4 copies) isolated from the fetal and maternal cells was subjected to PCR-based WGA, Ampli 1TM Whole Genome Amplification Kit (Silicon Biosystems) which provides balanced and complete amplification of the entire genome of a cell, following the manufacturer' s protocol.
• Each library was quantified by RT-PCR, using the KAPA library quantification kit (KAPA Biosystems - Illumina).
• the DNA standards provided in the kit represent a 10-fold dilution series from 0.0002 to 20 pM. Two nM of each individual library were combined for multiplex sequencing, which eliminated sequencing lane effects.
• the libraries were sequenced in a rapid flow cell on an Illumina HiSeq 2500.
• Variants shared between each maternal and fetal pair were identified, followed by filtering out of shared homozygotes and shared heterozygotes, using a variant allele percentage >85 to be considered a homozygote, and a variant allele percentage ⁇ 85 to be considered a heterozygote. Secondly, the minimal difference between the maternal and fetal variant allele percentage was set at 25. The remaining variants were considered informative, i.e. maternal homozygote/fetal heterozygote, or maternal heterozygote/fetal homozygote.
• Chromosomal coverage ratios were determined by calculating the mean coverage value per chromosome for each targeted region as a ratio of diploid chromosome coverage. [00205] Sequencing data, shown in Tables VI and VII, revealed coverage of all chromosomes.
• Figure 4 is a Cercos heat map that indicates mean coverage by targeted regions of all exome sequences on a logio scale, indicating each chromosome (1-22, X, Y) in maternal (M) and fetal (F) samples. Higher coverages are indicated by darker gray color.
• the outermost circle represents the chromosome ideograms with the pter-qter orientation in a clockwise direction and the centromeres in black.
• the expanded region (left) shows detailed coverage for X and Y chromosomes.
• Figure 5A shows the relative coverage of autosomal, X and Y chromosomes after WGA for the fetal and maternal cells
• Figure 5B shows the relative coverage of autosomal, X and Y chromosomes after WGA for newborn blood samples (blood spots) and maternal blood samples, which are used as reference standards.
• the dots in Figures 5 A and 5B represent individual data points; Boxes: 25th to 75th percentiles; horizontal lines within the boxes: median; whiskers: 1.5 x Inter Quartile Range (3rd quartile - 1st quartile).
• the expected ratio of autosomal:X:Y chromosomes in maternal DNA (2:2:0) was obtained (1.96 + 0.03 to 2.29 + 0.46 to 0.05 + 0.08), while the expected ratio for a male fetus (2: 1: 1) was obtained (1.67 + 0.11 to 1.16 + 0.24 to 2.46 + 1.02) in trophoblast DNA from isolated fetal nuclei, see Figure 5A.
• the elevated proportion of Y chromosome in trophoblast cells was most likely due to incorrect alignment, because much of the Y chromosome is shared with the X chromosome.
• WGA amplified DNA see Figure 5A, showed larger variations in chromosomal coverage than unamplified DNA, see Figure 5B, due to preferential amplification of non-GC-rich regions during WGA.
• Fetal DNA from isolated nuclei of fetal trophoblast cells from endocervical samples or maternal endocervical cells isolated from endocervical samples was sequenced and compared to sequenced reference fetal DNA isolated from matched placental specimens. Table VIII. Overview of samples and results for targeted sequencing
• the percentage of libraries between 200-1000 bp was calculated based on the library traces and was mixed accordingly at the core facility.
• a pool size of 200-1 kb was selected, and the size- selected negative controls were mixed into the pool.
• the samples were sequenced on a MiSeq FGX system including positive and negative controls (Illumina).
• ForenSeq Targeted sequencing data was extracted from the ForenSeq Universal Analysis software, which provides a run quality report and detailed genotype. From this report, data was extracted and allele percentages determined, using the read depth of each Short Tandem Repeat (STR) or Single Nucleotide Polymorphism (SNP). For STR data, the two dominant alleles with highest relative peaks were used for further analysis.
• STR Short Tandem Repeat
• SNP Single Nucleotide Polymorphism
• Maternal and placental SNP profiles were then compared, and the non- informative SNPs (i.e., identical maternal and placental alleles) were filtered out.
• the informative SNPs the deviations obtained from the maternal sample were subtracted from the fetal sample and multiplied by two (to account for two maternal alleles contributing to the fetal sample), yielding the percentage of maternal contamination.
• each autosomal STR was used. The dominant alleles with highest relative peaks were summed, to establish the median and range, which discriminated the pure ( ⁇ 15% contamination) from impure samples. Pure samples had median STR alleles >85%, while impure samples had median STR alleles ⁇ 80%. To establish a cutoff for discriminating pure from impure samples, 82.5% was chosen.
• the adjacent mini-plots show distribution of shared STRs (upper mini-plot) and discriminatory STRs (middle and lower mini-plots) with the percentage of reads indicated numerically and by shade intensity for each dominant allele.
• Item 1 A method of isolating DNA of a fetus of an ongoing pregnancy, comprising: obtaining a maternal endocervical sample containing maternal cells and fetal extravillous trophoblast cells from a pregnant subject; isolating fetal extravillous trophoblast cells from the maternal endocervical sample, producing isolated fetal extravillous trophoblast cells contaminated with maternal DNA; lysing the isolated fetal extravillous trophoblast cells; isolating fetal nuclei from the lysed fetal extravillous trophoblast cells, producing isolated fetal nuclei, thereby removing at least a portion of the contaminating maternal DNA; lysing the isolated fetal nuclei; and purifying genomic DNA from the isolated fetal nuclei, producing purified fetal genomic DNA.
• Item 2 The method of item 1, further comprising treating the isolated fetal nuclei to remove at least a portion of the
• Item 3 The method of item 1 or 2, further comprising treating the isolated fetal nuclei with a DNAse prior to lysing the isolated fetal nuclei, thereby removing at least a portion of the contaminating maternal DNA.
• Item 4 The method of any of items 1-3, further comprising treating the isolated extravillous trophoblast cells with a DNAse prior to isolating fetal extravillous trophoblast cells from the maternal endocervical sample, thereby removing at least a portion of the contaminating maternal DNA.
• Item 5 The method of any of items 1-4, further comprising treating the isolated extravillous trophoblast cells with a DNAse prior to lysing isolated fetal extravillous trophoblast cells, thereby removing at least a portion of the contaminating maternal DNA.
• Item 6 The method of any of items 1-5, wherein the fetal extravillous trophoblast cells are fixed prior to treating the fetal extravillous trophoblast cells with the DNAse and the DNAse is attached to a support, preventing entry of the DNAse into the fetal extravillous trophoblast cells.
• Item 7 The method of any of items 1-5, wherein the fetal extravillous trophoblast cells are not fixed prior to treating the fetal extravillous trophoblast cells with the DNAse.
• Item 8 The method of any of items 1-7, wherein the purified fetal genomic DNA is characterized by a fetal fraction in the range of 10%-100%.
• Item 9 The method of any of items 1-8, wherein the purified fetal genomic DNA is characterized by a fetal fraction in the range of 25%-100%.
• Item 10 A method of assaying DNA of a fetus of an ongoing pregnancy, comprising: obtaining a maternal endocervical sample containing fetal extravillous trophoblast cells from a pregnant subject; isolating fetal extravillous trophoblast cells from the maternal endocervical sample; lysing the isolated fetal extravillous trophoblast cells; isolating fetal nuclei from the lysed fetal extravillous trophoblast cells; lysing the isolated fetal nuclei and purifying genomic DNA from the isolated fetal nuclei; and assaying the purified fetal genomic DNA, thereby determining a characteristic of a genomic DNA sequence of the purified fetal genomic DNA, and thereby assaying genomic DNA of a fetus of an ongoing pregnancy.
• Item 11 The method of item 10, wherein assaying the purified fetal genomic DNA determines a characteristic of at least one individual nucleotide in a genomic DNA sequence of the purified fetal genomic DNA, thereby assaying DNA of a fetus of an ongoing pregnancy with single base resolution.
• Item 12 The method of item 10 or 11, wherein the assaying comprises a method selected from the group consisting of: sequencing, high resolution melt analysis, methylation analysis, capillary electrophoresis, mass spectrometry, single strand conformation polymorphism, single base extension, restriction fragment length polymorphism.
• Item 13 The method of any of items 10-12, wherein the sequencing comprises a method selected from the group consisting of: massively parallel signature sequencing, single-molecule real-time sequencing, polony sequencing, ion semiconductor, pyrosequencing, sequencing by synthesis, sequencing by ligation and chain termination sequencing.
• Item 14 The method of any of items 10-13, further comprising treating the isolated fetal nuclei to remove at least a portion of the contaminating maternal DNA.
• Item 15 The method of any of items 10-14, further comprising treating the isolated fetal nuclei with a DNAse prior to lysing the isolated fetal nuclei, thereby removing at least a portion of the contaminating maternal DNA.
• Item 16 The method of any of items 10-15, further comprising treating the isolated extravillous trophoblast cells with a DNAse prior to isolating fetal extravillous trophoblast cells from the maternal endocervical sample, thereby removing at least a portion of the contaminating maternal DNA.
• Item 17 The method of any of items 10-16, further comprising treating the isolated extravillous trophoblast cells with a DNAse prior to lysing isolated fetal extravillous trophoblast cells, thereby removing at least a portion of the contaminating maternal DNA.
• Item 18 The method of any of items 10-17, wherein the fetal extravillous trophoblast cells are fixed prior to treating the fetal extravillous trophoblast cells with the DNAse and the DNAse is attached to a support, preventing entry of the DNAse into the fetal extravillous trophoblast cells.
• Item 19 The method of any of items 10-17, wherein the fetal extravillous trophoblast cells are not fixed prior to treating the fetal extravillous trophoblast cells with the DNAse.
• Item 20 The method of any of items 10-19, wherein the purified fetal genomic DNA is characterized by a fetal fraction in the range of 10%- 100%.
• Item 21 The method of any of items 10-20, wherein the purified fetal genomic DNA is characterized by a fetal fraction in the range of 25%-100%.
• Item 22 A method of isolating DNA of a fetus of an ongoing pregnancy substantially as described herein.
• Item 23 A method of assaying DNA of a fetus of an ongoing pregnancy with single base resolution substantially as described herein.
• compositions and methods described herein are presently representative of preferred embodiments, exemplary, and not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art. Such changes and other uses can be made without departing from the scope of the invention as set forth in the claims.

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