Development of the real tiime PCR method for SNP typing of the blood types

1.建立了AS-TaqMan聚合酶链式反应(AS-TaqMan)、MBG探针TaqMan聚合酶链式反应(TaqMan)、熔融曲线分析和错配扩增分析等方法。1.建立了AS-TaqMan聚合酶链式反应(AS-TaqMan)和SYBR Green聚合酶链式反应(SYBR Green)方法，用于检测糖蛋白A(GypA)、低密度脂蛋白受体(LDLR)、血红蛋白G(HBGG)、D7S8和群特异性成分(GC)等位基因。各基因座上不同基因型间的阈值循环数(Ct)差异有统计学意义。对Gypa进行了进一步的分析，因为它表明血糖素A与MN血型有很强的相关性。建立了MN血型AS-TaqMan和SYBR Green两种基因分型方法。2.建立了ABO基因分型的TaqMan-MBG杂交法。用TaqMan聚合酶链式反应检测ABO糖基转移酶基因第703、796、930位碱基缺失。3.建立了快速检测ABO糖基转移酶基因第261位碱基缺失、793位碱基替换、930位碱基替换的杂交探针方法。设计了两个荧光标记的杂交探针，通过熔融曲线分析进行缺失和替换的检测。4.建立了错配扩增MN基因分型方法和熔化曲线分析方法。错配扩增分析涉及在实时聚合酶链式反应中具有不同结合效率的错配的聚合酶链式反应。此外，还发展了熔化曲线分析的MN基因分型方法。所开发的实时荧光聚合酶链式反应方法简单、快速、准确，适合于高通量应用。

Real-time PCR methods using AS-TaqMan PCR, TaqMan PCR with MBG probe, melting curve analyses and mismatch amplification assay were developed.1.I have developed AS-TaqMan PCR assay and SYBR Green PCR assay for detecting of glycophorin A (GYPA), low density lipoprotein receptor (LDLR), hemoglobin G (HBGG), D7S8 and group specific component (GC) alleles which are typed by AmpliType PM + OQA1 PCR Amplification and Typing. The differences of threshold cycles (Ct) values between different genotypes on each of the loci were statistically differed significantly. Further analysis was carried out about GYPA, because it shows a strong correlation between glycophorin A and MN blood group. Then, AS-TaqMan PCR assay and SYBR Green PCR assay for genotyping of MN blood group have developed.2.TaqMan PCR with MBG hybridization probe for ABO genotyping has developed. The base deletion at 261^<st> base substitutions at 703^<rd>, 796^<th> and 930^<th> of cDNA of ABO glycosyltransferase were detected by TaqMan PCR with MBG probe.3.I have developed the hybridization probe assay to rapidly detect the base deletion at 261^<st> and base substitutions at 793^<rd> and 930^<th> of cDNA of ABO glycosyltransferase. Two fluorescent-labeled hybridization probe were designed and detection of deletion and substitutions were performed by melting curve analyses.4.MN genotyping by mismatch amplification assay and melting curve analyses have developed. Mismatch amplification assay involves mismatched PCR primers that have different binding efficiencies within a real time PCR. MN genotyping by melting curve analyses has also developed.Developed real-time PCR assays are simple, rapid, and accurate, as well as suitable for high-throughput applications.