Haplotype-contained PCR Products Analysis By Sequencing With Selective Restriction Of Primer Extension

Currently,haplotype analysis is an important medium for linkage disequilibrium analysis,finding pathogenic genes,and conducting chromosome structure studies.However,existing methods for haplotyping,such as single-molecule dilution,cloning,and allele-specific polymerase chain reaction,are more cumbersome and expensive than genotyping.We develop a strategy for PCR products haplotype analysis of diploid organism.After PCR products including two SNP loci genotyped,the haplotypes were determined when individuals were homozygous at one or both loci.When individuals were heterozygous at both loci,the possible haplotypes had two combinations–meaning that there was a need for further analysis.PCR products heterozygous at both loci were first sequenced with allele-specific primers.The primers that matched one of the alleles at the 3? end were applied to determine the haplotype directly.But some allele-specific primers were nonspecifically extended which resulted in false results,alternatively specific dideoxyribonucleoside triphosphate(dd NTP)was added so as to selectively block sequencing primers during sequencing assays.If the added dd NTP was complementary with DNA templates,the sequencing primers were blocked.If not,the unblocked primers would continue to extend,thus making haplotypes be determined by pyrosequencing or Sanger sequencing.To validate its feasibility,UGT1A1*6 and UGT1A1*28 related to Gilbert's syndrome and K1637 K and S1647 T related to Parkinson's disease were selected as experimental sites.Haplotypes of PCR products,including UGT1A1*6 and UGT1A1*28,were successfully analyzed by Sangersequencing with allele-specific primers.Also,haplotypes of PCR products,including K1637 K and S1647 T,could not be determined by Sanger sequencing with allele-specific primers but successfully analyzed by pyrosequencing with dd NTP-blocked primers.As a result,this method is able to effectively haplotype two adjacent heterozygous in PCR products.It is simple,fast,and irrespective of short read length of pyrosequencing.Overall,we fully hope it will provide a new and precise technology to identify haplotypes of conventional PCR products in clinical samples.