The invention provides a method useful for detection of genetic disorders. The method comprises determining the sequence of alleles of a locus of interest, and quantitating a ratio for the alleles at the locus of interest, wherein the ratio indicates the presence or absence of a chromosomal abnormality. The present invention also provides a non-invasive method for the detection of chromosomal abnormalities in a fetus. The invention is especially useful as a non-invasive method for determining the sequence of fetal DNA. The invention further provides methods of isolation of free DNA from a sample.

Description of the Invention

BRIEF SUMMARY OF THE INVENTION

The invention is directed to a method for detection of genetic disorders including mutations and chromosomal abnormalities. In some embodiments, the present invention is used to detect mutations, and chromosomal abnormalities including but not limited to translocation, transversion, monosomy, trisomy, and other aneuploidies, deletion, addition, amplification, fragment, translocation, and rearrangement. Numerous abnormalities can be detected simultaneously. The present invention also provides a non-invasive method to determine the sequence of fetal DNA from a sample of a pregnant female. The present invention can be used to detect any alternation in gene sequence as compared to the wild type sequence including but not limited to point mutation, reading frame shift, transition, transversion, addition, insertion, deletion, addition-deletion, frame-shift, missense, reverse mutation, and microsatellite alteration. The present invention also provides a method for isolating free nucleic acid from a sample containing nucleic acid. The present invention also provides compositions and kits.

In one aspect, the invention is directed to methods for detecting chromosomal abnormalities. In one embodiment, the present invention is directed to a method for detecting chromosomal abnormalities, said method comprising quantitating the relative amount of the alleles at a heterozygous locus of interest, where the heterozygous locus of interest was previously identified by determining the sequence of alleles at a locus of interest from template DNA, wherein said relative amount is expressed as a ratio, and wherein said ratio indicates the presence or absence of a chromosomal abnormality.

In some embodiments, determining the sequence includes using a method that is allele specific PCR, mass spectrometry, hybridization, primer extension, fluorescence resonance energy transfer (FRET), sequencing, Sanger dideoxy sequencing, DNA microarray, GeneCHIP arrays, HuSNP arrays, CodeLink Arrays, BeadArray Technology, MassARRAY, MassEXTEND, SNP-IT, TaqMan, InvaderStrand Assay, southern blot, slot blot, dot blot, or MALDI-TOF mass spectrometry.

In some embodiments, template DNA is obtained from human, non-human, mammal, reptile, cattle, cat, dog, goat, swine, pig, monkey, ape, gorilla, bull, cow, bear, horse, sheep, poultry, mouse, rat, fish, dolphin, whale, or shark. In an embodiment, the template DNA is obtained from a human source. In a preferred embodiment, the template DNA is obtained from a pregnant human female. In some embodiments, the template DNA is obtained from a sample that is a cell, fetal cell, tissue, blood, serum, plasma, saliva, urine, tear, vaginal secretion, sweat, umbilical cord blood, chorionic villi, amniotic fluid, embryonic tissue, an embryo, a two-celled embryo, a four-celled embryo, an eight celled embryo, a 16-celled embryo, a 32-celled embryo, a 64-celled embryo, a 128-celled embryo, a 256-celled embryo, a 512-celled embryo, a 1024-celled embryo, lymph fluid, cerebrospinal fluid, mucosa secretion, peritoneal fluid, ascitic fluid, fecal matter, or body exudates. In these embodiments, the sample may be mixed with an agent that inhibits cell lysis to inhibit the lysis of cells, if cells are present, where the agent is a membrane stabilizer, a cross-linker, or a cell lysis inhibitor. In some of these embodiments, agent is a cell lysis inhibitor, and may be glutaraldehyde, derivatives of glutaraldehyde, formaldehyde, formalin, or derivatives of formaldehyde. In some of these embodiments the sample is blood and in one embodiment the sample is blood from a pregnant female, e.g., a human female. In the latter embodiment, the fetus may be at a gestational age selected from the group consisting of: 0-4, 4-8, 8-12, 12-16, 16-20, 20-24, 24-28, 28-32, 32-36, 36-40, 40-44, 44-48, 48-52, or more than 52 weeks. In some of these embodiments, the template DNA may be obtained from plasma or from serum from the blood. In these embodiments, the template DNA may include a mixture of maternal DNA and fetal DNA, and in one embodiment, prior to determining the sequence of alleles of a locus of interest from template DNA, maternal DNA is sequenced to identify a homozygous locus of interest, and the homozygous locus of interest is the locus of interest analyzed in the template DNA. In another embodiment, maternal DNA is sequenced to identify a heterozygous locus of interest, and the heterozygous locus of interest is the locus of interest analyzed in the template DNA.

In embodiments, alleles of multiple loci of interest are sequenced and their relative amounts quantitated and expressed as a ratio. In one embodiment, the sequence of alleles of one to tens to hundreds to thousands of loci of interest on a single chromosome on template DNA is determined. In another embodiment, the sequence of alleles of one to tens to hundreds to thousands of loci of interest on multiple chromosomes is determined.

In an embodiment, the locus of interest is suspected of containing a single nucleotide polymorphism or mutation. The method can be used for determining sequences of multiple loci of interest concurrently. The template DNA can comprise multiple loci from a single chromosome. The template DNA can comprise multiple loci from different chromosomes. The loci of interest on template DNA can be amplified in one reaction. Alternatively, each of the loci of interest on template DNA can be amplified in a separate reaction. The amplified DNA can be pooled together prior to digestion of the amplified DNA. Each of the labeled DNA containing a locus of interest can be separated prior to determining the sequence of the locus of interest. In one embodiment, at least one of the loci of interest is suspected of containing a single nucleotide polymorphism or a mutation.

There is no limitation as to the chromosomes that can be compared. The ratio for the alleles at a heterozygous locus of interest on any chromosome can be compared to the ratio for the alleles at a heterozygous locus of interest on any other chromosome. In another embodiment, the ratio of alleles at a heterozygous locus of interest on a chromosome is compared to the ratio of alleles at a heterozygous locus of interest on two, three, four or more than four chromosomes. In another embodiment, the ratio of alleles at multiple loci of interest on a chromosome is compared to the ratio of alleles at multiple loci of interest on two, three, four, or more than four chromosomes. In embodiments, the ratio for alleles at heterozygous loci of interest on a chromosome are summed and compared to the ratio for alleles at heterozygous loci of interest on a different chromosome, where a difference in ratios indicates the presence of a chromosomal abnormality. In some of these embodiments, the chromosomes that are compared are human chromosomes such as chromosome 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, X, or Y. In one of the latter embodiments, the ratio for the alleles at heterozygous loci of interest of chromosomes 13, 18, and 21 are compared. In another embodiment, the sequence of one to tens to hundreds to thousands of loci of interest on the template DNA obtained from a sample of a pregnant female is determined. In one embodiment, the loci of interest are on one chromosome. In another embodiment, the loci of interest are on multiple chromosomes.

In some embodiments, determining the sequence of the alleles comprises amplifying alleles of a locus of interest on a template DNA using a first and a second primer, where the second primer contains a recognition site for a restriction enzyme such that digestion with the restriction enzyme generates a 5' overhang containing the locus of interest; digesting the amplified DNA with the restriction enzyme that recognizes the recognition site on the second primer; incorporating a nucleotide into the digested DNA by using the 5' overhang containing the locus of interest as a template; and determining the sequence of the alleles of the locus of interest by determining the sequence of the DNA into which the nucleotide was incorporated. In one embodiment, determination of the sequence of the locus of interest in comprises detecting a nucleotide.

In other embodiments, determining the sequence of alleles comprises amplifying alleles of a locus of interest on a template DNA using a first and second primers, where the second primer contains a recognition site for a restriction enzyme such that digestion with the restriction enzyme generates a 5' overhang containing the locus of interest; digesting the amplified DNA with the restriction enzyme that recognizes the recognition site on the second primer; incorporating nucleotides into the digested DNA of (b), where a nucleotide that terminates elongation, and is complementary to the locus of interest of an allele, is incorporated into the 5' overhang of said allele, and a nucleotide complementary to the locus of interest of a different allele is incorporated into the 5' overhang of said different allele, and said terminating nucleotide, which is complementary to a nucleotide in the 5' overhang of said different allele, is incorporated into the 5' overhang of said different allele; and determining the sequence of the alleles of a locus of interest by determining the sequence of the DNA into which the complementary nucleotides have been incorporated. In one embodiment, determination of the sequence of the locus of interest comprises detecting a nucleotide.

The incorporation of a nucleotide may be accomplished by a DNA polymerase, including but not limited to E. coli DNA polymerase, Klenow fragment of E. coli DNA polymerase I, T7 DNA polymerase, T4 DNA polymerase, T5 DNA polymerase, Klenow class polymerases, Taq polymerase, bacteriophage 29, REDTaq.TM. Genomic DNA polymerase, Pfu DNA polymerase, Vent DNA polymerase or sequenase. Incorporation of a nucleotide may include incorporation of a labeled nucleotide, or labeled and unlabeled nucleotides. One nucleotide, two nucleotides, three nucleotides, four nucleotides, five nucleotides, or more than five nucleotides can be incorporated. A combination of labeled and unlabeled nucleotides can be incorporated. The labeled nucleotide may be a dideoxynucleotide triphosphate (also referred to as "dideoxy") or deoxynucleotide triphosphate (also referred to as "deoxy"). The unlabeled nucleotide may be a dideoxynucleotide triphosphate or deoxynucleotide triphosphate. Labeled nucleotides may be labeled with a radioactive molecule, fluorescent molecule, antibody, antibody fragment, hapten, carbohydrate, biotin, derivative of biotin, phosphorescent moiety, luminescent moiety, electrochemiluminescent moiety, chromatic moiety, and moiety having a detectable electron spin resonance, electrical capacitance, dielectric constant or electrical conductivity. In one embodiment, the labeled nucleotide is labeled with a fluorescent molecule. The incorporation of a fluorescent labeled nucleotide may further comprise using a mixture of fluorescent and unlabeled nucleotides.

In one embodiment, the determination of the sequence of the locus of interest comprises detecting the incorporated nucleotide. The detection method includes but is not limited to gel electrophoresis, capillary electrophoresis, microchannel electrophoresis, polyacrylamide gel electrophoresis, fluorescence detection, fluorescence polarization, DNA sequencing, Sanger dideoxy sequencing, ELISA, mass spectrometry, time of flight mass spectrometry, quadrupole mass spectrometry, magnetic sector mass spectrometry, electric sector mass spectrometry, fluorometry, infrared spectrometry, ultraviolet spectrometry, palentiostatic amperometry, DNA hybridization, DNA microarray, GeneChip arrays, HuSNP arrays, BeadArrays, MassExtend, SNP-IT, TaqMan assay, Invader assay, MassCleave, southern blot, slot blot, or dot blot.

In embodiments, first and second primers contain a portion of a restriction enzyme recognition site that contains a variable nucleotide, where the full restriction enzyme recognition site is generated after amplification. In some embodiments, the 3' region of said primers can contain mismatches with the template DNA, and digestion with said restriction enzyme generates a 5' overhang containing the locus of interest. In some embodiments, the restriction enzyme recognition site is for a restriction enzyme that includes but is not limited to BsaJ I, Bssk I, Dde I, EcoN I, Fnu4H I, Hinf I, or ScrF I. In some embodiments, the restriction enzyme cuts DNA at a distance from the recognition site. In some of these embodiments, the recognition site is for a Type IIS restriction enzyme. In some of these embodiments, the Type IIS restriction enzyme includes but is not limited to Alw I, Alw26 I, Bbs I, Bbv I, BceA I, Bmr I, Bsa I, Bst71 I, BsmA I, BsmnB I, BsmF I, BspM I, Ear I, Fau I, Fok I, Hga I, Ple I, Sap I, SSfaN I, or Sthi32 I.

In some embodiments, the recognition site for restriction enzymes includes but is not limited to BsaJ I (5'C.sup..dwnarw.CNNGG 3'), BssK I (5'.sup..dwnarw.CCNGG 3'), Dde I (5'C.sup..dwnarw.TNAG 3'), EcoN I (5'CCTNN.sup..dwnarw.NNNAGG 3' (SEQ ID NO: 7)), Fnu4H I (5'GC.sup..dwnarw.NGC 3'), Hinf I (5'G.sup..dwnarw.ANTC 3'), PflF 1(5' GACN.sup..dwnarw.NNGTC 3'), Sau96 I (5' G.sup..dwnarw.GNCC 3'), ScrF I (5'CC.sup..dwnarw.NGG 3'), Tthl 11 I (5' GACN.sup..dwnarw.NNGTC 3'), and more preferably Fnu4H I and EcoN I, is generated after amplification.

The first and/or second primer can contain a tag at the 5' terminus. In some embodiments, the first primer contains a tag at the 5' terminus. The tag can be used to separate the amplified DNA from the template DNA. The tag can be used to separate the amplified DNA containing the labeled nucleotide from the amplified DNA that does not contain the labeled nucleotide. The tag can be any chemical moiety including but not limited to radioisotope, fluorescent reporter molecule, chemiluminescent reporter molecule, antibody, antibody fragment, hapten, biotin, derivative of biotin, photobiotin, iminobiotin, digoxigenin, avidin, enzyme, acridinium, sugar, enzyme, apoenzyme, homopolymeric oligonucleotide, hormone, ferromagnetic moiety, paramagnetic moiety, diamagnetic moiety, phosphorescent moiety, luminescent moiety, electrochemiluminescent moiety, chromatic moiety, moiety having a detectable electron spin resonance, electrical capacitance, dielectric constant or electrical conductivity, or combinations thereof. In some embodiments, the tag is biotin. The biotin tag is used to separate amplified DNA from the template DNA using a streptavidin matrix. The streptavidin matrix may be coated on wells of a microtiter plate.

In some embodiments, the annealing length of the second primer is selected from the group consisting of 35-30, 30-25, 25-20, 20-15, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, and less than 4 bases.

In embodiments, the method of amplification includes but is not limited to polymerase chain reaction, self-sustained sequence reaction, ligase chain reaction, rapid amplification of cDNA ends, polymerase chain reaction and ligase chain reaction, Q-beta phage amplification, strand displacement amplification, or splice overlap extension polymerase chain reaction. In some embodiments, the method of amplification is PCR. In some of these embodiments, an annealing temperature for cycle 1 of PCR is about the melting temperature of the portion of the 3' region of the second primer that anneals to the template DNA. In some of the latter embodiments, an annealing temperature for cycle 2 of PCR is about the melting temperature of the portion of the 3' region of the first primer that anneals to the template DNA. In some of the latter embodiments, an annealing temperature for the remaining cycles of PCR is at about the melting temperature of the entire second primer.

In another aspect, the invention provides methods of determining the sequence of a locus of interest from a sample comprising free fetal DNA, where an agent that inhibits cell lysis has been added to the sample to inhibit lysis of cells, if cells are present, where the agent is a membrane stabilizer, a cross-linker, or a cell lysis inhibitor.

In some embodiments, the agent is a cell lysis inhibitor, and in some of these embodiments, the cell lysis inhibitor includes but is not limited to glutaraldehyde, derivatives of glutaraldehyde, formaldehyde, derivatives of formaldehyde, or formalin. In embodiments, the sample includes but is not limited to tissue, cell, blood, serum, plasma, urine, or vaginal secretion. In some embodiments, the sample is blood. In some of these embodiments, the template DNA is isolated from the serum, in other embodiments the template DNA is isolated from plasma. In some embodiments, the sample contains free maternal template DNA and free fetal template DNA. In some embodiments, prior to determining the sequence, template DNA was isolated. In some embodiments, prior to determining the sequence of the locus of interest on fetal DNA, the sequence of the locus of interest on maternal template DNA was determined. In some embodiments, prior to determining the sequence of the locus of interest on fetal DNA, the sequence of the locus of interest on paternal template DNA was determined. In some embodiments, the locus of interest is a single nucleotide polymorphism. In other embodiments, the locus of interest is a mutation. In some embodiments, the sequence of multiple loci of interest is determined. In some of these embodiments, the multiple loci of interest are on multiple chromosomes.

In some embodiments, the sequence is determined by: (a) amplifying a locus of interest on a template DNA using a first and second primers, where the second primer contains a recognition site for a restriction enzyme such that digestion with the restriction enzyme generates a 5' overhang containing the locus of interest; (b) digesting the amplified DNA with the restriction enzyme that recognizes the recognition site on the second primer; (c) incorporating a nucleotide into the digested DNA of (b) by using the 5' overhang containing the locus of interest as a template; and (d) determining the sequence of the locus of interest by determining the sequence of the DNA of (c).

In other embodiments, the sequence is determined by: (a) amplifying alleles of a locus of interest on a template DNA using a first and second primers, where the second primer contains a recognition site for a restriction enzyme such that digestion with the restriction enzyme generates a 5' overhang containing the locus of interest; (b) digesting the amplified DNA with the restriction enzyme that recognizes the recognition site on the second primer; (c) incorporating nucleotides into the digested DNA of (b), where a nucleotide that terminates elongation, and is complementary to the locus of interest of an allele, is incorporated into the 5' overhang of said allele, and a nucleotide complementary to the locus of interest of a different allele is incorporated into the 5' overhang of said different allele, and the terminating nucleotide, which is complementary to a nucleotide in the 5' overhang of said different allele, is incorporated into the 5' overhang of said different allele; and (d) determining the sequence of the alleles of a locus of interest by determining the sequence of the DNA of (c).

In some embodiments, the restriction enzyme cuts DNA at a distance from the recognition site. In some of these embodiments, the recognition site includes but is for a Type IIS restriction enzyme, for example Alw I, Alw26 I, Bbs I, Bbv I, BceA I, Bmr I, Bsa I, Bst71 I, BsmA I, BsmB I, BsmF I, BspM I, Ear I, Fau I, Fok I, Hga I, Ple I, Sap I, SSfaN I, or Sthi32 I.

In some embodiments, the method of amplification maybe, for example, polymerase chain reaction, self-sustained sequence reaction, ligase chain reaction, rapid amplification of cDNA ends, polymerase chain reaction and ligase chain reaction, Q-beta phage amplification, strand displacement amplification, or splice overlap extension polymerase chain reaction. In some embodiments, the method of amplification is by PCR. In some of these embodiments, an annealing temperature for cycle 1 of PCR is about the melting temperature of the portion of the 3' region of the second primer that anneals to the template DNA. In some of the latter embodiments, an annealing temperature for cycle 2 of PCR is about the melting temperature of the portion of the 3' region of the first primer that anneals to the template DNA. In some of the latter embodiments, an annealing temperature for the remaining cycles of PCR is at about the melting temperature of the entire second primer.

In some embodiments, the sequence of a locus of interest was determined using allele specific PCR, mass spectrometry, hybridization, primer extension, fluorescence polarization, fluorescence resonance energy transfer (FRET), fluorescence detection, sequencing, Sanger dideoxy sequencing, DNA microarray, southern blot, slot blot, dot blot, or MALDI-TOF mass spectrometry.

In some embodiments, the sequence of a locus of interest is determined by (1) amplification of the locus of interest; (2) hybridization of amplified loci to GeneCHIP array (3) washing GeneCHIP array; (4) staining the GeneCHIP array with detectable reagents; and (5) scanning GeneCHIP array. In some of these embodiments, the amplification method in (1) is polymerase chain reaction, self-sustained sequence reaction, ligase chain reaction, rapid amplification of cDNA ends, polymerase chain reaction and ligase chain reaction, Q-beta phage amplification, strand displacement amplification, or splice overlap extension polymerase chain reaction. In some embodiments, the method of amplification is by PCR. In some embodiments, the staining method comprises streptavidin phycoeryhthrin and biotinylated anti-streptavidin. In some embodiments, an agent that inhibits cell lysis has been added to the sample to inhibit the lysis of cells, if present, where the agent is membrane stabilizer, cross-linker, or cell lysis inhibitor. In some embodiments, the agent is a cell lysis inhibitor. In some of these embodiments, the cell lysis inhibitor is formalin at a percentage selected from the group consisting of: 0.0001-0.03%, 0.03-0.05%, 0.05-0.08%, 0.08-0.1%, 0.1-0.3%, 0.3-0.5%, 0.5-0.7%, 0.7-0.9%, 0.9-1.2%, 1.2-1.5%, 1.5-2%, or 2-3%. In some embodiments, the concentration of formalin in the sample is 0.1%.

In some embodiments, the sequence of a locus of interest is determined by (1) amplification of the locus of interest; (2) amplicon fragmentation; (3) hybridization of fragmented amplicons to CodeLink Arrays; (4) extension reaction to incorporate a nucleotide; and (5) detection of incorporated nucleotides. In some of these embodiments, the amplification method is polymerase chain reaction, self-sustained sequence reaction, ligase chain reaction, rapid amplification of cDNA ends, polymerase chain reaction and ligase chain reaction, Q-beta phage amplification, strand displacement amplification, or splice overlap extension polymerase chain reaction. In some embodiments, the method of amplification is by PCR. In some embodiments, the amplicon fragmentation is by exonuclease digestion. In some embodiments, the incorporated nucleotide is a dideoxynucleotide or deoxynucleotide. In some embodiments, the incorporated nucleotide is labeled with a radioactive molecule, fluorescent molecule, antibody, antibody fragment, hapten, carbohydrate, biotin, derivative of biotin, phosphorescent moiety, luminescent moiety, electrochemiluminescent moiety, chromatic moiety, and moiety having a detectable electron spin resonance, electrical capacitance, dielectric constant or electrical conductivity. In some embodiments, the labeled nucleotide is labeled with a fluorescent molecule. In some embodiments, an agent that inhibits cell lysis has been added to the sample to inhibit the lysis of cells, if present, where the agent is membrane stabilizer, cross-linker, or cell lysis inhibitor. In some embodiments, the agent is a cell lysis inhibitor. In some of these embodiments, the cell lysis inhibitor is formalin at a percentage selected from the group consisting of: 0.0001-0.03%, 0.03-0.05%, 0.05-0.08%, 0.08-0.1%, 0.1-0.3%, 0.3-0.5%, 0.5-0.7%, 0.7-0.9%, 0.9-1.2%, 1.2-1.5%, 1.5-2%, or 2-3%. In some embodiments, the concentration of formalin in the sample is 0.1%.

In some embodiments, the sequence of a locus of interest is determined by using BeadArray Technology. In some embodiments, an agent that inhibits cell lysis has been added to the sample to inhibit the lysis of cells, if present, where the agent is membrane stabilizer, cross-linker, or cell lysis inhibitor. In some embodiments, the agent is a cell lysis inhibitor. In some of these embodiments, the cell lysis inhibitor is formalin at a percentage selected from the group consisting of: 0.0001-0.03%, 0.03-0.05%, 0.05-0.08%, 0.08-0.1%, 0.1-0.3%, 0.3-0.5%, 0.5-0.7%, 0.7-0.9%, 0.9-1.2%, 1.2-1.5%, 1.5-2%, or 2-3%. In some embodiments, the concentration of formalin in the sample is 0.1%.

In some embodiments, the sequence of a locus of interest is determined by (1) amplification of the locus of interest; (2) dephosphorylation of the unused reagents in (1); (3) in vitro transcription reaction of the products of (2); (4) RNase A cleavage of the products of (3); (5) mixing the products of (4) with CleanResin; (6) transfer products of (5) to SpectroCHIP; and (7) analysis of the SpectroCHIP. In some of these embodiments, the amplification method is polymerase chain reaction, self-sustained sequence reaction, ligase chain reaction, rapid amplification of cDNA ends, polymerase chain reaction and ligase chain reaction, Q-beta phage amplification, strand displacement amplification, or splice overlap extension polymerase chain reaction. In some embodiments, the method of amplification is by PCR. In some embodiments, the dephosphorylation reaction is catalyzed by shrimp alkaline phosphatase. In some embodiments, an agent that inhibits cell lysis has been added to the sample to inhibit the lysis of cells, if present, where the agent is membrane stabilizer, cross-linker, or cell lysis inhibitor. In some embodiments, the agent is a cell lysis inhibitor. In some of these embodiments, the cell lysis inhibitor is formalin at a percentage selected from the group consisting of: 0.0001-0.03%, 0.03-0.05%, 0.05-0.08%, 0.08-0.1%, 0.1-0.3%, 0.3-0.5%, 0.5-0.7%, 0.7-0.9%, 0.9-1.2%, 1.2-1.5%, 1.5-2%, or 2-3%. In some embodiments, the concentration of formalin in the sample is 0.1%.

In some embodiments, the sequence of a locus of interest is determined by (1) amplification of a locus of interest; (2) dephosphorylation of the unused reagents in (1); (3) hybridization of a primer to the locus of interest; (4) incorporation of a nucleotide; (5) mixing the products of (4) with CleanResin; (6) transfer products of (5) to SpectroCHIP; and (7) analysis of the SpectroCHIP. In some of these embodiments, the amplification method is polymerase chain reaction, self-sustained sequence reaction, ligase chain reaction, rapid amplification of cDNA ends, polymerase chain reaction and ligase chain reaction, Q-beta phage amplification, strand displacement amplification, or splice overlap extension polymerase chain reaction. In some embodiments, the method of amplification is by PCR. In some embodiments, the dephosphorylation reaction is catalyzed by shrimp alkaline phosphatase. In some embodiments, hybridization of primer is adjacent to the locus of interest. In some embodiments, the incorporated nucleotide is a dideoxynucleotide or deoxynucleotide. In some embodiments, the incorporated nucleotide is labeled with radioactive molecule, fluorescent molecule, antibody, antibody fragment, hapten, carbohydrate, biotin, derivative of biotin, phosphorescent moiety, luminescent moiety, electrochemiluminescent moiety, chromatic moiety, and moiety having a detectable electron spin resonance, electrical capacitance, dielectric constant or electrical conductivity. In some embodiments, the labeled nucleotide is labeled with a fluorescent molecule. In some embodiments, an agent that inhibits cell lysis has been added to the sample to inhibit the lysis of cells, if present, where the agent is membrane stabilizer, cross-linker, or cell lysis inhibitor. In some embodiments, the agent is a cell lysis inhibitor. In some of these embodiments, the cell lysis inhibitor is formalin at a percentage selected from the group consisting of: 0.0001-0.03%, 0.03-0.05%, 0.05-0.08%, 0.08-0.1%, 0.1-0.3%, 0.3-0.5%, 0.5-0.7%, 0.7-0.9%, 0.9-1.2%, 1.2-1.5%, 1.5-2%, or 2-3%. In some embodiments, the concentration of formalin in the sample is 0.1%.

In some embodiments, the sequence of a locus of interest is determined by (1) amplification of the locus of interest; (2) exonuclease treatment of the products of (1); (3) single stranded DNA of (2) is annealed to an oligonucleotide; (4) incorporation of a nucleotide using the annealed template and primer of (3); (5) detection of the incorporated nucleotide. In some embodiments, the amplification method is by polymerase chain reaction, self-sustained sequence reaction, ligase chain reaction, rapid amplification of cDNA ends, polymerase chain reaction and ligase chain reaction, Q-beta phage amplification, strand displacement amplification, or splice overlap extension polymerase chain reaction. In some embodiments, the method of amplification is by PCR. In some embodiments, the primer hybridizes adjacent to the locus of interest. In some embodiment, the incorporated nucleotide is a dideoxynucleotide or deoxynucleotide. In some embodiments, the incorporation reaction comprises two terminating nucleotides and two non-terminating nucleotides. In some embodiments, the incorporated nucleotide is labeled with radioactive molecule, fluorescent molecule, antibody, antibody fragment, hapten, carbohydrate, biotin, derivative of biotin, phosphorescent moiety, luminescent moiety, electrochemiluminescent moiety, chromatic moiety, and moiety having a detectable electron spin resonance, electrical capacitance, dielectric constant or electrical conductivity. In some embodiments, the terminating nucleotides are labeled with radioactive molecule, fluorescent molecule, antibody, antibody fragment, hapten, carbohydrate, biotin, derivative of biotin, phosphorescent moiety, luminescent moiety, electrochemiluminescent moiety, chromatic moiety, and moiety having a detectable electron spin resonance, electrical capacitance, dielectric constant or electrical conductivity. In some embodiments, the labeled nucleotide is labeled with a fluorescent molecule. In some embodiments, the terminating nucleotides are labeled with a fluorescent molecule. In some embodiments, an agent that inhibits cell lysis has been added to the sample to inhibit the lysis of cells, if present, where the agent is membrane stabilizer, cross-linker, or cell lysis inhibitor. In some embodiments, the agent is a cell lysis inhibitor. In some of these embodiments, the cell lysis inhibitor is formalin at a percentage selected from the group consisting of: 0.0001-0.03%, 0.03-0.05%, 0.05-0.08%, 0.08-0.1%, 0.1-0.3%, 0.3-0.5%, 0.5-0.7%, 0.7-0.9%, 0.9-1.2%, 1.2-1.5%, 1.5-2%, or 2-3%. In some embodiments, the concentration of formalin in the sample is 0.1%.

In some embodiments, the sequence of a locus of interest is determined by (1) amplification of the locus of interest, wherein the amplification reaction comprises a forward primer, a reverse primer, and a probe that anneals to the locus of interest, which is within the region of the amplicon; and (2) detection of the PCR products, wherein the amount of PCR product is used to determine the presence or absence of a specific genetic sequence. In some embodiments, the amplification is by PCR. In some embodiments, the probe contains a reporter dye at the 5' end and the 3' end contains a quenching dye. In some embodiments, the PCR products are detected using the ABI 7700 Sequence Detection System. In some embodiments, an agent that inhibits cell lysis has been added to the sample to inhibit the lysis of cells, if present, where the agent is membrane stabilizer, cross-linker, or cell lysis inhibitor. In some embodiments, the agent is a cell lysis inhibitor. In some of these embodiments, the cell lysis inhibitor is formalin at a percentage selected from the group consisting of: 0.0001-0.03%, 0.03-0.05%, 0.05-0.08%, 0.08-0.1%, 0.1-0.3%, 0.3-0.5%, 0.5-0.7%, 0.7-0.9%, 0.9-1.2%, 1.2-1.5%, 1.5-2%, or 2-3%. In some embodiments, the concentration of formalin in the sample is 0.1%.

In another aspect, the invention provides methods for determining the sequence of a locus of interest in a sample containing fetal DNA.

In some embodiments, the method for determining the sequence includes (a) amplifying a locus of interest on a template DNA using a first and second primers, where the second primer contains a recognition site for a restriction enzyme such that digestion with the restriction enzyme generates a 5' overhang containing the locus of interest; (b) digesting the amplified DNA with the restriction enzyme that recognizes the recognition site on the second primer; (c) incorporating a nucleotide into the digested DNA of (b) by using the 5' overhang containing the locus of interest as a template; and (d) determining the sequence of the locus of interest by determining the sequence of the DNA of (c).

In other embodiments, the method for determining the sequence includes (a) amplifying alleles of a locus of interest on a template DNA using a first and second primers, where the second primer contains a recognition site for a restriction enzyme such that digestion with the restriction enzyme generates a 5' overhang containing the locus of interest; (b) digesting the amplified DNA with the restriction enzyme that recognizes the recognition site on the second primer; (c) incorporating nucleotides into the digested DNA of (b), where a nucleotide that terminates elongation, and is complementary to the locus of interest of an allele, is incorporated into the 5' overhang of said allele, and a nucleotide complementary to the locus of interest of a different allele is incorporated into the 5' overhang of said different allele, and said terminating nucleotide, which is complementary to a nucleotide in the 5' overhang of said different allele, is incorporated into the 5' overhang of said different allele; and (d) determining the sequence of the alleles of a locus of interest by determining the sequence of the DNA of (c).

In embodiments, the sample is cell, tissue, blood, serum, plasma, saliva, urine, tears, vaginal secretion, sweat, umbilical cord blood, chorionic villi, amniotic fluid, embryonic tissue, embryo, a two-celled embryo, a four-celled embryo, an eight-celled embryo, a 16-celled embryo, a 32-celled embryo, a 64-celled embryo, a 128-celled embryo, a 256-celled embryo, a 512-celled embryo, a 1024-celled embryo, lymph fluid, cerebrospinal fluid, mucosa secretion, peritoneal fluid, ascitic fluid, fecal matter, or body exudates.

In another aspect, the invention provides methods for preparing a sample for analysis that include isolating free nucleic acid from a sample that contains nucleic acid, where an agent that inhibits cell lysis has been added to the sample to inhibit lysis of cells, if cells are present, where the agent is membrane stabilizer, cross-linker, or cell lysis inhibitor. In this aspect, the portion of the sample that is to be analyzed is the free nucleic acid, not the cellular portion. In an embodiment, the present invention provides a method for isolating nucleic acid said method comprising (a) obtaining a sample containing nucleic acid; (b) adding a cell lysis inhibitor, cell membrane stabilizer, or cross-linker to the sample of (a); and (c) isolating nucleic acid. In an embodiment, the method is used for isolating free nucleic acid. In an embodiment, the method is used for isolating free fetal nucleic acid. In another embodiment, the present invention provides a method for isolating free fetal nucleic acid said method comprising (a) obtaining a sample containing nucleic acid; (b) adding a cell lysis inhibitor, cell membrane stabilizer, or cross-linker to the sample of (a); (c) isolating the plasma from the blood sample, wherein the plasma is isolated by centrifuging the blood sample; and (d) removing the supernatant, which contains the plasma, using procedures to minimize disruption of the "buffy-coat."

In some embodiments, the agent is cell lysis inhibitor, and in some of these embodiments, the cell lysis inhibitor is glutaraldehyde, derivatives of glutaraldehyde, formaldehyde, formalin, and derivatives of formaldehyde, crosslinkers, primary amine reactive crosslinkers, sulfhydryl reactive crosslinkers, sulfhydryl addition or disulfide reduction, carbohydrate reactive crosslinkers, carboxyl reactive crosslinkers, photoreactive crosslinkers, cleavable crosslinkers, AEDP, APG, BASED, BM(PEO).sub.3, BM(PEO).sub.4, BMB, BMDB, BMH, BMOE, BS3, BSOCOES, DFDNB, DMA, DMP, DMS, DPDPB, DSG, DSP, DSS, DST, DTBP, DTME, DTSSP, EGS, HBVS, sulfo-BSOCOES, Sulfo-DST, Sulfo-EGS or compounds listed in Table XXIII (see Original Patent).

In some embodiments the cell lysis inhibitor is formalin. In some of these embodiments, the final concentration of formalin in the sample is 0.0001-0.03%, 0.03-0.05%, 0.05-0.08%, 0.08-0.1%, 0.1-0.3%, 0.3-0.5%, 0.5-0.7%, 0.7-0.9%, 0.9-1.2%, 1.2-1.5%, 1.5-2%, or 2-3%. In one embodiment, the final concentration of formalin in the sample is 0.1%.

An agent that stabilizes cell membranes may be added to the sample including but not limited to aldehydes, urea formaldehyde, phenol formaldehyde, DMAE (dimethylaminoethanol), cholesterol, cholesterol derivatives, high concentrations of magnesium, vitamin E, and vitamin E derivatives, calcium, calcium gluconate, taurine, niacin, hydroxylamine derivatives, bimoclomol, sucrose, astaxanthin, glucose, amitriptyline, isomer A hopane tetral phenylacetate, isomer B hopane tetral phenylacetate, citicoline, inositol, vitamin B, vitamin B complex, cholesterol hemisuccinate, sorbitol, calcium, coenzyme Q, ubiquinone, vitamin K, vitamin K complex, menaquinone, zonegran, zinc, ginkgo biloba extract, diphenylhydantoin, perftoran, polyvinylpyrrolidone, phosphatidylserine, tegretol, PABA, disodium cromglycate, nedocromil sodium, phenyloin, zinc citrate, mexitil, dilantin, sodium hyaluronate, or polaxamer 188.

In another embodiment, an agent that prevents DNA destruction is added to the sample including but not limited to DNase inhibitors, zinc chloride, ethylenediaminetetraacetic acid, guanidine-HCI, guanidine isothiocyanate, N-lauroylsarcosine, and Na-dodecylsulphate.

In some embodiments, the sample is obtained from human, non-human, mammal, reptile, cattle, cat, dog, goat, swine, pig, monkey, ape, gorilla, bull, cow, bear, horse, sheep, poultry, mouse, rat, fish, dolphin, whale, or shark. In some of these embodiments, the sample is obtained from a human source.

In some embodiments, the sample containing nucleic acid is obtained from any nucleic acid containing source including but not limited to a cell, fetal cell, tissue, blood, serum, plasma, saliva, urine, tear, vaginal secretion, breast fluid, breast milk, sweat, umbilical cord blood, chorionic villi, amniotic fluid, embryonic tissue, embryo, a two-celled embryo, a four-celled embryo, an eight-celled embryo, a 16-celled embryo, a 32-celled embryo, a 64-celled embryo, a 128-celled embryo, a 256-celled embryo, a 512-celled embryo, a 1024-celled embryo lymph fluid, cerebrospinal fluid, mucosa secretion, peritoneal fluid, ascitic fluid, fecal matter, or body exudates. In some of these embodiments, the sample is blood.

In embodiments the sample is from a pregnant female. In an embodiment, the sample is obtained from a pregnant human female. In an embodiment, the sample is blood obtained from a pregnant female and, e.g., the nucleic acid is isolated from plasma obtained from blood of a pregnant female; the plasma is generated using procedures designed to minimize the amount of maternal cell lysis. In some of these embodiments, the blood is obtained from a human pregnant female when the fetus is at a gestational age of 0-4, 4-8, 8-12, 12-16, 16-20, 20-24, 24-28, 28-32, 32-36, 36-40, 40-44, 44-48, 48-52, or more than 52 weeks. In some of these embodiments, the sample is obtained from plasma from the blood.

In some embodiments, the isolation of nucleic acid includes a centrifugation step; e.g., in some embodiments free nucleic acid is isolated from plasma obtained from blood, for example from a pregnant female. In some embodiments, the centrifugation step is performed with the centrifuge braking power set to zero (the centrifuge comes to a stop by natural deceleration). In some embodiments, the centrifugation step is performed at a speed of 0-50 rpm, 50-100 rpm, 100-200 rpm, 200-300 rpm, 300-400 rpm, 400-500 rpm, 500-600 rpm, 600-700 rpm, 700-800 rpm, 800-900 rpm, 900-1000 rpm, 1000-2000 rpm, 2000-3000 rpm, 3000-4000 rpm, 4000-5000 rpm, 5000-6000 rpm, 6000-7000 rpm, 7000-8000 rpm, or greater than 8000 rpm. In one embodiment, the blood, e.g., from the pregnant female, is centrifuged at a speed less than 4000 rpm. In another embodiment, the acceleration power of the centrifuge is not used.

In another aspect, the invention provides a method for detecting a chromosomal abnormality by (a) determining the sequence of alleles of a locus of interest from template DNA, and (b) quantitating the relative amount of the alleles at a heterozygous locus of interest that was identified from the locus of interest of (a), wherein said relative amount is expressed as a ratio, and wherein said ratio indicates the presence or absence of a chromosomal abnormality.

In yet another aspect, the invention provides compositions.

In one embodiment, the invention provides a composition containing fetal DNA and maternal DNA, where the percentage of free fetal DNA in the total free DNA of the composition is about 15-16% fetal DNA, about 16-17% fetal DNA, about 17-18% fetal DNA, about 18-19% fetal DNA, about 19-20% fetal DNA, about 20-21% fetal DNA, about 21-22% fetal DNA, about 22-23% fetal DNA, about 23-24% fetal DNA, about 24-25% fetal DNA, about 25-35% fetal DNA, about 35-45% fetal DNA, about 45-55% fetal DNA, about 55-65% fetal DNA, about 65-75% fetal DNA, about 75-85% fetal DNA, about 85-90% fetal DNA, about 90-91% fetal DNA, about 91-92% fetal DNA, about 92-93% fetal DNA, about 93-94% fetal DNA, about 94-95% fetal DNA, about 95-96% fetal DNA, about 96-97% fetal DNA, about 97-98% fetal DNA, about 98-99% fetal DNA, or about 99-99.7% fetal DNA.

In another embodiment, the invention provides a composition containing fetal DNA and maternal DNA, where the percentage of free fetal DNA in the total free DNA of the composition is about 15-16% fetal DNA, about 16-17% fetal DNA, about 17-18% fetal DNA, about 18-19% fetal DNA, about 19-20% fetal DNA, about 20-21% fetal DNA, about 21-22% fetal DNA, about 22-23% fetal DNA, about 23-24% fetal DNA, about 24-25% fetal DNA, about 25-35% fetal DNA, about 35-45% fetal DNA, about 45-55% fetal DNA, about 55-65% fetal DNA, about 65-75% fetal DNA, about 75-85% fetal DNA, about 85-90% fetal DNA, about 90-91% fetal DNA, about 91-92% fetal DNA, about 92-93% fetal DNA, about 93-94% fetal DNA, or about 94-95% fetal DNA.

In yet another aspect, the invention provides a prenatal diagnostic method including analyzing a composition comprising fetal DNA and maternal DNA, where the percentage of free fetal DNA in the total free DNA of the composition is about 15-16% fetal DNA, about 16-17% fetal DNA, about 17-18% fetal DNA, about 18-19% fetal DNA, about 19-20% fetal DNA, about 20-21% fetal DNA, about 21-22% fetal DNA, about 22-23% fetal DNA, about 23-24% fetal DNA, about 24-25% fetal DNA, about 25-35% fetal DNA, about 35-45% fetal DNA, about 45-55% fetal DNA, about 55-65% fetal DNA, about 65-75% fetal DNA, about 75-85% fetal DNA, about 85-90% fetal DNA, about 90-91% fetal DNA, about 91-92% fetal DNA, about 92-93% fetal DNA, about 93-94% fetal DNA, or about 94-95% fetal DNA.

In still yet another aspect, the invention provides a kit for use in any of the methods of the invention, where the kit contains a set of primers used in the method, where the second primer contains a sequence that generates a recognition site for a restriction enzyme such that digestion with the restriction enzyme generates a 5'-overhang containing the locus of interest, and a set of instructions.
 

Claim 1 of 140 Claims

1. A method for detecting the presence or absence of a fetal chromosomal abnormality, said method comprising: quantitating a ratio of the relative amount of alleles at a heterozygous locus of interest in a mixture of template DNA, wherein said mixture comprises maternal DNA and fetal DNA, and wherein said mixture of maternal DNA and fetal DNA has been obtained from a sample from a pregnant female, and further wherein said heterozygous locus of interest has been identified by determining the sequence of alleles at the locus of interest, and wherein said ratio indicates the presence or absence of a fetal chromosomal abnormality.

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