DHPLC Applications:Finding DNA variation on the Y chromosome

Denaturing High-Performance Liquid Chromatogr- aphy (DHPLC) is a recently developed technique for the detection of single nucleotide polymorphisms (SNPs) and mutations. It involves the comparison between two or more DNAs as a mixture of denatured and reannealed PCR products. The methodology is based on the principle of reversed phase liquid chromatography and uses a unique DNA separation matrix. The exquisite sensitivity of the technique is determined by adjusting the oven temperature. Elution of DNA fragments is dependant on the chain length and sequence and can be predicted by computation. Under partially denaturing conditions heteroduplices formed upon mixing, denaturing, and reannealing of two, or more, chromosomes that differ in sequence, are retained less than their corresponding homodupulices and sequence variation is recognized by the appearance of two, or more, peaks in the chromatographs. Numerous SNPs have been identified on the non-recombinant portion of the Y chromosome by using this technique. To investigate the DNA variation within Pakistan 15 Y-SNPs, an Alu insertion, a LINE1 insertion and the 12f2 deletion, mapping on the non-recombining portion of the human Y chromosome, were typed in 834 Pakistani males. The combination of these biallelic markers identified 11 stable Y chromosomal lineages or Y haplogroups in the Pakistani population. Haplogroup frequencies were generally similar to those in neighboring geographical areas and indicate that there was a common pool of Y lineages within Pakistan that are predominantly from West and Central Asia.: