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US 2004/0106102 A1Published application
Rapid analysis of variations in a genome
AssigneeRavgen, Inc.
InventorRavinder S. Dhallan
Life of this patent
Estimating the term…
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.
Claims70
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).
3 more elements
A method for determining a sequence of alleles of a locus of interest, said method comprising:
- (a) amplifying a locus of interest on a template DNA using a first and second primers, wherein the second primer contains a portion of a recognition site for a restriction enzyme, wherein a full recognition site for the restriction enzyme is generated upon amplification of the template DNA 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 full recognition site generated by the second primer and the template DNA;
- (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.
- determining the sequence of the locus of interest by determining the sequence of the DNA of (c).
3 more elements
A method for determining a sequence of alleles of a locus of interest, said method comprising:
- (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 that cuts DNA at a distance from the recognition site and digestion with the restriction enzyme generates a 5′ overhang containing the locus of interest, and wherein the first primer contains a recognition site for a restriction enzyme that is different from the recognition site for the restriction enzyme on the second primer and contains a tag at the 5′ end;
- (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 labeled 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) digesting the DNA of (c) with the restriction enzyme that recognizes the recognition site on the first primer;
- and
- (e) determining the sequence of the alleles of a locus of interest by determining the sequence of the digested DNA of (d) containing the labeled nucleotide.
5 more elements
A method for determining a sequence of alleles of a locus of interest, said method comprising:
- (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 that cuts DNA at a distance from the recognition site and digestion with the restriction enzyme generates a 5′ overhang containing the locus of interest, wherein the first primer contains a recognition site for a restriction enzyme that is different from the recognition site for the restriction enzyme on the second primer, and contains a tag at the 5′ end, and wherein the annealing temperature for cycle 1 of PCR is at about the melting temperature of the portion of 3′ region of the second primer that anneals to the template DNA, the annealing temperature for cycle 2 of PCR is at about the melting temperature of the portion of the 3′ region of the first primer that anneals to the template DNA, and the annealing temperature for the remaining cycles is at about the melting temperature of the entire second primer;
- (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 labeled 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) digesting the DNA of (c) with the restriction enzyme that recognizes the recognition site on the first primer;
- and
- (e) determining the sequence of the alleles of a locus of interest by determining the sequence of the digested DNA of (d) containing the labeled nucleotide.
5 more elements
A method for determining a sequence of a locus of interest, said method comprising:
- (a) replicating a region of DNA comprising a locus of interest from a template polynucleotide by using a first and a second primer, wherein 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;
- (b) digesting the DNA with the restriction enzyme that recognizes the recognition site generated by the second primer to create a DNA fragment;
- (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).
2 more elements
A DNA fragment containing a locus of interest to be sequenced and a recognition site for a restriction enzyme, wherein digestion with the restriction enzyme creates a 5′ overhang on the DNA fragment, and wherein the locus of interest and the restriction enzyme recognition site are in relationship to each other such that digestion with the restriction enzyme generates a 5′ overhang containing the locus of interest.
Dependencies are inferred from the claim text.