Restriction enzyme genotyping
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, 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 having the selected polymorphic site within an overhanging end;
(c) treating the nucleic acid fragment generated in step (b) to convert the overhanging end to a blunt end by extension of the recessed portion of the overhanging end; and
(d) analyzing one or both strands of the nucleic acid fragment generated in step (c) 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
12 Claims
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1. A method for determining the nucleotide present at a selected polymorphic site in a target nucleic acid molecule, 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 having the selected polymorphic site within an overhanging end; (c) treating the nucleic acid fragment generated in step (b) to convert the overhanging end to a blunt end by extension of the recessed portion of the overhanging end; and (d) analyzing one or both strands of the nucleic acid fragment generated in step (c) to identify the nucleotide present at the selected polymorphic site. - View Dependent Claims (3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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2. A method for determining the nucleotide present at a selected polymorphic site in a target nucleic acid molecule, 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, wherein the cleavage site of the second restriction enzyme is such that the polymorphic site within the nucleic acid fragment is present within an overhanging end; (b) digesting the amplification product with the second restriction enzyme in the presence of an enzyme that converts the overhanging end to a blunt end by extension of the recessed portion of the overhanging end under conditions which permit multiple cycles of digestion and conversion such that an oligonucleotide containing the polymorphic site is generated; and (c) analyzing the oligonucleotide to identify the nucleotide present at the selected polymorphic site.
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Specification