Patent attributes
The present invention is directed to a method and kit for determining the molecular haplotype of a gene in a diploid DNA sample. The method discriminates between two haplotypes on the basis of a difference of one or more nucleotides using allele-specific PCR amplification in combination with long range PCR, using a DNA polymerase enzyme having 3′→5′ exonuclease activity, using annealing temperature conditions sufficiently greater than the predicted annealing temperature (Tm) to effect selective hybridization and extension of an allele-specific extension primer to the target allele relative to the variant allele. The present invention is particularly useful, for example, to determine the haplotype of a gene having multi-allelic genetic loci separated by a distance in which accurate PCR amplification of a single fragment containing the multiple genetic loci cannot be performed without using a DNA polymerase enzyme having 3′→5′ exonuclease activity, generally about 5 kilobases or more.
