Methods and compositions for nucleotide analysis
First Claim
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1. A method for identifying at least one target nucleotide within a target sequence comprising:
- generating multiple fragments of the target sequence by digesting the target sequence with at least one restriction enzyme;
amplifying the multiple fragments using a first set of primers to generate multiple amplified fragments, wherein the first set of primers comprises a first primer and a second primer;
amplifying at least one subset of the multiple amplified fragments to generate at least one amplified subset of the multiple amplified fragments using a first set of extended primers, wherein the first set of extended primers comprises a first extended primer which comprises the sequence of the first primer and one additional nucleotide on the 3′
end and a second extended primer which comprises the sequence of the second primer and one additional nucleotide on the 3′
end; and
identifying the at least one target nucleotide within a target sequence by identifying the at least one target nucleotide in at least one of the amplified subsets of the multiple amplified fragments.
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Abstract
The invention relates generally to the field of nucleic acid sequence analysis. In certain embodiments, the analysis is genotyping. In certain embodiments, the analysis involves detecting single nucleotide polymorphisms (SNPs). The invention also relates to methods, kits, and computer software for nucleic acid analysis.
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Citations
12 Claims
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1. A method for identifying at least one target nucleotide within a target sequence comprising:
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generating multiple fragments of the target sequence by digesting the target sequence with at least one restriction enzyme;
amplifying the multiple fragments using a first set of primers to generate multiple amplified fragments, wherein the first set of primers comprises a first primer and a second primer;
amplifying at least one subset of the multiple amplified fragments to generate at least one amplified subset of the multiple amplified fragments using a first set of extended primers, wherein the first set of extended primers comprises a first extended primer which comprises the sequence of the first primer and one additional nucleotide on the 3′
end and a second extended primer which comprises the sequence of the second primer and one additional nucleotide on the 3′
end; and
identifying the at least one target nucleotide within a target sequence by identifying the at least one target nucleotide in at least one of the amplified subsets of the multiple amplified fragments. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
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11. A kit for the amplification of amplified fragment length polymorphisms comprising:
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a first set of primers comprising a first primer and a second primer used to generate amplified fragment length polymorphisms; and
a first set of extended primers comprising a third extended primer which comprises the sequence of the first extended primer and one additional nucleotide on the 3′
end and a fourth extended primer which comprises the sequence of the second extended primer and one additional nucleotide on the 3′
end.
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12. A method for identifying a nucleotide at a SNP site comprising:
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digesting genomic DNA with two restriction enzymes to generate multiple fragments;
ligating an adaptor to each end of the multiple fragments of the target sequence;
amplifying the multiple fragments to generate multiple amplified fragments using a first set of primers, wherein the first set of primers comprises a first primer and a second primer, wherein the first primer comprises a sequence complementary to the adaptor on one end of the multiple fragments of the target sequence and the second primer comprises a sequence complementary to the adaptor on the other end of the multiple fragments of the target sequence. adding a first set of extended primers, wherein the first set of extended primers comprises a first extended primer which comprises the sequence of the first primer and one additional nucleotide on the 3′
end and a second extended primer which comprises the sequence of the second primer and one additional nucleotide on the 3′
end;
amplifying a subset of the multiple amplified fragments using the first set of extended primers to generate a first amplified subset of the multiple amplified fragments;
adding a second set of extended primers, wherein the second set of extended primers comprises a third extended primer which comprises the sequence of the first extended primer and one additional nucleotide on the 3′
end and a fourth extended primer which comprises the sequence of the second extended primer and one additional nucleotide on the 3′
end;
amplifying a second subset of the multiple amplified fragments employing the second set of extended primers to generate a second amplified subset of the multiple amplified fragments;
performing an asymmetric amplification of the second amplified subset of the multiple amplified fragments to generate multiple single stranded fragments;
performing a single base extension reaction using at least one of the multiple single stranded fragments, a single base extension primer, and sequence terminators having an indicator specific for the specific nucleotide of the sequence terminator, wherein the single base extension primer comprises (i) at its 3′
end a sequence complementary to nucleotides immediately adjacent to the SNP site and (ii) a unique sequence identifier, to obtain a single base extension product; and
identifying the nucleotide at the SNP site from the specific indicator.
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Specification