文献_2012-A new genotyping method for detecting low abundance single nucleotide mutations.pdfVIP

Cell Biochem Biophys (2012) 62:161–167 DOI 10.1007/s12013-011-9277-2 ORIGINAL PAPER A New Genotyping Method for Detecting Low Abundance Single Nucleotide Mutations Based on Gap Ligase Chain Reaction and Quantitative PCR Assay Ping Yi • Hongmei Jiang • Li Li • Faguo Dai • Yingru Zheng • Jian Han • Zhuqin Chen • Jianxin Guo Published online: 18 October 2011 Springer Science+Business Media, LLC 2011 Abstract We tested applicability of a new genotyping Introduction technique to detect a low abundance CD17 (A ? T) mutation of b-globin gene. The technique utilized a com- Recent studies found that free fetal DNA exists in maternal bined gap ligase chain reaction (Gap-LCR) and quantita- plasma during pregnancy [ 1, 2]. These findings highlight tive PCR (qPCR) methods. One pair of Gap-LCR primers new opportunities for non-invasive prenatal diagnosis was modified by adding specific sequences to the 50 end of using maternal blood. The technical challenge, however, is the upstream and the 30 end of the downstream primer that fetal DNA is presented in small quantities in maternal which served as a combining sequence for qPCR. First, blood and that fragments of maternal allele can interfere specific mutation is detected using Gap-LCR; then, ligation with detection of fetal DNA. Therefore, detection of a low products are detected by qPCR. Our results show that the abundance fetal DNA requires utilization of highly sensi- amount of LCR products is directly proportional to the tive and specific techniques [3]. DNA ligase has a strong amount of template DNA. We further demonstrate that this ability to identify a single nucleotide mutation and is, technique detects a low abundance mutant DNA with a ther