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

建立了检测血型糖蛋白A（GYPA）、低密度脂蛋白受体（LDLR）、血红蛋白G（HBGG）、低密度脂蛋白受体（LDLR）的AS-TaqMan PCR和SYBR绿色PCR方法，并对检测结果进行了分析。D 7S 8和组特异性组分（GC）等位基因，通过AmpliType PM + OQA 1 PCR扩增和分型进行分型。各位点不同基因型间的阈值循环（Ct）值差异均达到显著水平。对GYPA进行了进一步的分析，因为它显示血型糖蛋白A和MN血型之间的强相关性。建立了MN血型基因分型的AS-TaqMan PCR法和SYBR绿色PCR法。2.建立了MBG杂交探针TaqMan PCR法ABO血型基因分型方法。建立<st><rd><th><th>了ABO糖基转移酶cDNA第261位碱基缺失、<st>第793位和<rd>第930位<th>碱基替换的探针杂交法。设计了两种荧光标记的杂交探针，并通过熔解曲线分析进行缺失和替换的检测。4.建立了错配扩增法和熔解曲线分析法用于MN基因分型。错配扩增测定涉及在真实的时间PCR内具有不同结合效率的错配PCR引物。通过熔解曲线分析的MN基因分型也得到了发展。开发的实时PCR检测方法简单、快速、准确，并且适合于高通量应用。

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.