Haplotype-Contained PCR Products Analysis by Sequencing with Selective Restriction of Primer Extension

Figure 1

(a) Haplotype analysis flow diagram of PCR products sequenced by selective restriction of primer extension. First, single-stranded PCR products including two SNP loci were genotyped by pyrosequencing, and samples that were heterozygous at both loci were selected for further haplotype analysis. Allele-specific primers were then applied to haplotype by Sanger sequencing. If the samples were able to be sequenced with allele-specific primers, their haplotype could be determined. If not, they were further analyzed. Third, for the samples that could not be haplotyped with an allele-specific primer, specific ddNTP was added so as to selectively block sequencing primers during sequencing assays. (b) Principle of restricting the designated primer extension with ddNTP-blocked primers. A primer was first hybridized to a base at the front of the first heterozygous locus in the PCR products. When specific ddNTP was added, some of the primers were blocked during primer extension while others could not be blocked. The unblocked primers continued to extend. Finally, haplotypes were determined by pyrosequencing or Sanger sequencing.

(a)

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