Methods for detection of genetic disorders
First Claim
1. A method for detecting a chromosomal abnormality, said method comprising:
- quantitating the relative amount of the alleles at a heterozygous locus of interest, wherein said heterozygous locus of interest was 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.
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Abstract
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.
395 Citations
180 Claims
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1. A method for detecting a chromosomal abnormality, said method comprising:
- quantitating the relative amount of the alleles at a heterozygous locus of interest, wherein said heterozygous locus of interest was 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.
- View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 103, 105, 106, 107, 108, 110, 111, 112, 113, 114, 115, 116, 118, 120, 121, 122, 123, 125, 126, 127, 128, 129, 130, 131, 132, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 145, 146, 147, 148, 149, 150, 151, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171)
- 58. A method comprising determining the sequence of a locus of interest on free fetal DNA from a sample comprising free fetal DNA, wherein agent that inhibits cell lysis has been added to said sample to inhibit lysis of cells, if cells are present, wherein said agent is selected from the group consisting of membrane stabilizer, cross-linker, and cell lysis inhibitor.
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84. A method for determining the sequence of a locus of interest in a sample comprising fetal DNA, said method comprising:
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(a) amplifying a locus of interest on a template DNA using a first and second primers, wherein 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). - View Dependent Claims (86, 180)
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85. A method for determining the sequence of a locus of interest in a sample comprising fetal DNA, said method comprising:
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(a) amplifying alleles of a locus of interest on a template DNA using a first and second primers, wherein 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), wherein;
(i) 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(ii) 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.(d) determining the sequence of the alleles of a locus of interest by determining the sequence of the DNA of (c).
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- 87. A method for preparing a sample for analysis comprising isolating free nucleic acid from a sample that contains nucleic acid, wherein an agent that inhibits cell lysis inhibitor has been added to the sample to inhibit lysis of cells, if cells are present, and wherein said agent is selected from the group consisting of membrane stabilizer, cross-linker, and cell lysis inhibitor.
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152. A method for detecting a chromosomal abnormality, said method comprising:
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(a) determining the sequence of alleles of a locus of interest from template DNA, (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.
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153. A composition comprising fetal DNA and maternal DNA, wherein the percentage of free fetal DNA in the total free DNA of the composition is selected from the group consisting of:
- 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, and about 99-99.7% fetal DNA.
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154. A composition comprising fetal DNA and maternal DNA, wherein the percentage of free fetal DNA in the total free DNA of the composition is selected from the group consisting of:
- 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, and about 94-95% fetal DNA.
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155. A prenatal diagnostic method comprising analyzing a composition comprising fetal DNA and maternal DNA, wherein the percentage of free fetal DNA in the total free DNA of the composition is selected from the group consisting of:
- 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, and about 94-95% fetal DNA.
Specification