Haplotyping method for multiple distal nucleotide polymorphisms

Abstract

The invention relates to methods for detecting genetic polymorphisms in an organism, particularly to the detection of genetic polymorphisms that are due to multiple distal nucleotide polymorphisms within a gene. Methods are provided for determining the haplotype structure of a gene, or other contiguous DNA segment, having two or more nucleotide polymorphisms that are separated by kilobases of DNA. The methods involve the use of PCR amplification and DNA ligation to bring the nucleotide polymorphisms on a particular allele of the gene into close proximity to facilitate the determination of haplotype structure.

1 . A kit for determining whether a first NP and a second NP separated by at least 200 nucleotides are located on the same DNA molecule in a nucleic acid sample comprising: 
 (a) a first PCR primer capable of specifically annealing to a region of said DNA molecule adjacent to said first NP which is distal to said second NP;    (b) a second PCR primer capable of specifically annealing to the region of said DNA molecule adjacent to said second NP which is distal to said first NP;    (c) a polymerase enzyme capable of catalyzing the PCR amplification of a fragment of said DNA molecule between the annealing site of said first PCR primer and the annealing site said second PCR primer;    (d) ligase enzyme capable of catalyzing the ligation of a first end of said fragment to a second end of said fragment to form a circularized segment; and    (e) means for detecting the presence or absence of said first NP and said second NP on said circularized segment.    
     
     
         2 . The kit of  claim 1  further comprising a restriction enzyme, wherein said primers contain a restriction enzyme recognition site for said restriction enzyme.