Methods for detection of genetic disorders
First Claim
1. A method for determining the sequence of a locus of interest from template DNA, wherein the template DNA comprises a mixture of fetal DNA and maternal DNA and is obtained from a sample from a pregnant female, said method comprising:
- (a) amplifying a locus of interest on the 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).
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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.
118 Citations
21 Claims
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1. A method for determining the sequence of a locus of interest from template DNA, wherein the template DNA comprises a mixture of fetal DNA and maternal DNA and is obtained from a sample from a pregnant female, said method comprising:
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(a) amplifying a locus of interest on the 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 (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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15. A method for determining the sequence of alleles of a locus of interest from template DNA, wherein the template DNA comprises a mixture of fetal DNA and maternal DNA and is obtained from a sample from a pregnant female, 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). - View Dependent Claims (16, 17, 18, 19, 20, 21)
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Specification