Methods for genetic analysis of DNA using biased amplification of polymorphic sites

Abstract

Methods for determing 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.

What is claimed is:  
     
         1 . A method of detecting the presence of a variance in a target nucleic acid sequence, comprising 
 amplifying a template nucleic acid sequence to generate an amplification product, wherein said amplifying results in the insertion of a bifunctional restriction enzyme recognition site into said amplification product; and    analyzing said amplification product.    
     
     
         2 . The method of  claim 1 , wherein the bifunctional restriction enzyme recognition site is inserted by a primer.  
     
     
         3 . The method of  claim 2 , wherein the primer contains a 5′ end complementary to said template nucleic acid, a 3′ end complementary to said template and said recognition site comprises a bifunctional restriction enzyme recognition site.  
     
     
         4 . The method of  claim 2 , wherein the bifuntional restriction enzyme recognition site is inserted into the amplification product using a primer which results in the incorporation of a sequence of bases not present in or complementary to the template.  
     
     
         5 . The method of  claim 1  further comprising the step of cleaving the amplification product with one or more restriction enzymes.  
     
     
         6 . The method of  claim 5 , wherein the restriction enzymes comprise at least one type IIS restriction enzyme.  
     
     
         7 . A method of detecting the presence of a variance in a target nucleic acid molecule by mass spectrometry comprising: 
 a) proving said target nucleic acid molecule;    b) providing a first primer, wherein said first primer results in the insertion of a restriction enzyme recognition site in an amplification product;    c) providing a second primer which is 100% complementary to the target nucleic acid molecule;    d) amplifying a portion of the target nucleic acid using said primers to generate said amplification product;    e) e) cleaving amplification product with at least one restriction enzyme, a product of said cleaving being a nucleic acid fragment including the putative variance and being sufficiently small to be analyzed by mass spectrometry; and    f) analyzing said fragment by mass spectrometry.    
     
     
         8 . The method of  claim 7 , wherein the restriction enzyme recognition site is bifunctional.  
     
     
         9 . The method of  claim 8 , wherein the first primer comprises a 5′ end complementary to said target, a 3′ end complementary to said target, and said recognition site comprised said bifunctional restriction enzyme recognition site.  
     
     
         10 . The method of  claim 7 , wherein said restriction enzymes comprise at least one type IIS restriction enzyme.