Rapid analysis of variations in a genome
First Claim
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1. A method for determining a sequence of alleles of a locus of interest, said method comprising:
- (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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Abstract
The invention provides a method useful for determining the sequence of large numbers of loci of interest on a single or multiple chromosomes. The method utilizes an oligonucleotide primer that contains a recognition site for a restriction enzyme such that digestion with the restriction enzyme generates a 5′ overhang containing the locus of interest. The 5′ overhang is used as a template to incorporate nucleotides, which can be detected. The method is especially amenable to the analysis of large numbers of sequences, such as single nucleotide polymorphisms, from one sample of nucleic acid.
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Citations
42 Claims
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1. A method for determining a sequence of alleles of a locus of interest, 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 (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)
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Specification