Quantitative detection of single nucleotide polymorphisms for a pooled sample by a bioluminometric assay coupled with modified primer extension reactions (BAMPER)

摘要: A new method for SNP analysis based on the detection of pyrophosphate (PPi) is demonstrated, which capable detecting small allele frequency differences between two DNA pools genetic association studies other than typing. The specific primer extension reactions coupled with PPi detection. As specificity primer-directed not enough quantitative analysis, artificial mismatched bases are introduced into 3′-terminal regions primers as a way improving switching characteristics reactions. best position in such third from 3′-terminus. Contamination endogenous PPi, produces large background signal level was removed using PPase to degrade during sample preparation process. It possible accurately and quantitatively analyze SNPs set that correspond wild-type mutant segments. termini these at mutation positions. Various types were successfully analyzed. very determine frequencies low 0.02. reproducible difference can be determined. accurate detect meaningful among pooled samples. sensitive 14 amol ssM13 DNA. proposed seems promising terms realizing cost-effective, large-scale human testing system.