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We develop a strategy for haplotype analysis of PCR products that contained two adjacent heterozygous loci using sequencing with specific primers, allele-specific primers, and ddNTP-blocked primers. To validate its feasibility, two sets of PCR products, including two adjacent heterozygous SNPs,  UGT1A1 ⁎6 (rs4148323) and  UGT1A1 ⁎28 (rs8175347), and two adjacent heterozygous SNPs, K1637K (rs11176013) and S1647T (rs11564148), were analyzed. Haplotypes of PCR products, including  UGT1A1 ⁎6 and  UGT1A1 ⁎28, were successfully analyzed by Sanger sequencing with allele-specific primers. Also, haplotypes of PCR products, including K1637K and S1647T, could not be determined by Sanger sequencing with allele-specific primers but were successfully analyzed by pyrosequencing with ddNTP-blocked primers. As a result, this method is able to effectively haplotype two adjacent heterozygous PCR products. It is simple, fast, and irrespective of short read length of pyrosequencing. Overall, we fully hope it will provide a new promising technology to identify haplotypes of conventional PCR products in clinical samples.

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