Methods for genetic analysis of DNA to detect sequence variances
First Claim
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1. A method for determining the nucleotide present at a selected polymorphic site in a target nucleic acid molecule, the method comprising:
- a) amplifying a portion of the target nucleic acid molecule comprising the selected polymorphic site using a first primer and a second primer, the second primer containing a recognition site for a first restriction enzyme and a recognition site for a second restriction enzyme, to generate amplification product containing a recognition site for the first restriction enzyme and a recognition site for the second restriction enzyme such that digestion of the amplification product with the first restriction enzyme and the second restriction enzyme generates a nucleic acid fragment containing the selected polymorphic site;
b) digesting the amplification product with the first restriction enzyme and the second restriction enzyme to generate a nucleic acid fragment containing the selected polymorphic site within a 5′
single-stranded overhang;
c) filling the recessed end corresponding to the 5′
overhang with nucleotides wherein at least one of the nucleotides is bromodeoxuridine; and
d) analyzing the nucleic acid fragment to identify the nucleotide present at the selected polymorphic site.
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Abstract
Methods for determining genotypes and haplotypes of genes are described. Also described are single nucleotide polymorphisms and haplotypes in the ApoE gene and methods of using that information.
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Citations
5 Claims
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1. A method for determining the nucleotide present at a selected polymorphic site in a target nucleic acid molecule, the method comprising:
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a) amplifying a portion of the target nucleic acid molecule comprising the selected polymorphic site using a first primer and a second primer, the second primer containing a recognition site for a first restriction enzyme and a recognition site for a second restriction enzyme, to generate amplification product containing a recognition site for the first restriction enzyme and a recognition site for the second restriction enzyme such that digestion of the amplification product with the first restriction enzyme and the second restriction enzyme generates a nucleic acid fragment containing the selected polymorphic site; b) digesting the amplification product with the first restriction enzyme and the second restriction enzyme to generate a nucleic acid fragment containing the selected polymorphic site within a 5′
single-stranded overhang;c) filling the recessed end corresponding to the 5′
overhang with nucleotides wherein at least one of the nucleotides is bromodeoxuridine; andd) analyzing the nucleic acid fragment to identify the nucleotide present at the selected polymorphic site. - View Dependent Claims (2, 3, 4, 5)
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Specification