Linear allele-specific long-range amplification: a novel method of long-range molecular haplotyping

Abstract

Haplotypes have been repeatedly shown to be more powerful than collections of single-locus markers in gene-mapping studies. Various haplotyping methods including statistical estimation are employed but molecular haplotyping, the acquisition of information directly on physical DNA sequences, has been in demand for its accuracy and independence from family pedigrees. We investigated the allelic specificity of long-range PCR, which was successful for long-range haplotyping in recent reports, and found problems of initial mispriming and crossover amplification significantly confounded its application. Based on these observations, we designed a novel method based on linear amplification of a hemizygous DNA segment with a single phosphorothioate-modified oligonucleotide. Our results revealed, with a single nucleotide polymorphism as the discriminative marker, downstream haplotypes of 14-15 kb DNA segment could be confidently scored. With two rounds of the method and five single nucleotide polymorphisms, molecular haplotypes of 29.3 kb spanning the HCR and CDSN genes, two genes associated with the susceptibility of psoriasis, of 11 members, belonging to a CEPH family, were revealed. Clear Mendelian segregation of 35 highly heterozygous SNPs confirmed the accuracy of the method. Problems of low specificity associated with long-range PCR were not observed. The simplicity, along with long-sequence accessibility and feasibility of a single nucleotide difference as the discriminative marker indicated our method holds promise for future gene-mapping studies.

An efficient method for multi-locus molecular haplotyping

“…Other approaches rely upon limiting dilution to isolate (statistically) single DNA molecules, followed by amplification and genotyping of two or more loci (25–28). However, haplotypes assembled by these techniques rarely exceed 20–30 kb in length (29–31), never involve more than a few loci and the methods are often inefficient, since only a few of the highly dilute samples which are genotyped will prove to contain informative molecules. An exception is the use of the ‘polony’ method to genotype intact chromosomal DNA molecules dispersed in an acrylamide film (32).…”