Methods for genetic analysis of DNA to detect sequence variances
View Patent ↗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.
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.
2. The method of claim 1 wherein the second primer comprises at least one nucleotide sequence that is not present in the target nucleic acid molecule.
3. The method of claim 2 wherein the second primer comprises 5′ nucleotide sequence that is complementary to a first portion of the target nucleic acid molecule, a 3′ nucleotide sequence that is complementary to a second portion of the target nucleic acid molecule, and a nucleotide sequence that is not complementary to the target nucleic acid molecule.
4. The method of claim 1 wherein the first restriction enzyme is a type IIS restriction enzyme.
5. The method of claim 1 wherein the step of analyzing the nucleic acid fragment to identify the nucleotide present at the selected polymorphic site comprises subjecting the nucleic acid fragment to mass spectrometry.