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

血型 SNP 分型实时 PCR 方法的开发

• 批准号: 14570378
• 负责人: NATA Masayuki
• 金额: $ 1.47万
• 依托单位: TOHOKU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14570378/
• 关键词: allele specific TaqMan PCR; SYBR Green I; MN genotyping; ABO genotyping; MGB probe; TaqMan PCR; melting curve analyses; Mismatch primer; ABO式血液型; genotying; 5'nuclease活性; FAM; VIC; TaqMan-ASA法; poly marker; 糖転移酵素cDNA; MN式血液型

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.

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。

项目成果

Hashiyada M, Funayama M, Nata M, Mimasaka S, Adachi N: "Discrimination of skeletal remains using mitochondrial DNA"Acta Crim Japon. 68. 129-134 (2002)

Hashiyada M、Funayama M、Nata M、Mimasaka S、Adachi N：“使用线粒体 DNA 区分骨骼遗骸”Acta Crim Japon。

Nata M, Hashiyada M: "Rapid detection of GYPA, LDLR, HBGG, D7S8 and GC alleles by real-time fluorescence PCR"International Congress series 1239. Progress in Forensic Genetics. 9. 27-32 (2002)

Nata M、Hashiyada M：“通过实时荧光 PCR 快速检测 GYPA、LDLR、HBGG、D7S8 和 GC 等位基因”国际大会系列 1239。法医遗传学进展。

Uehara S, Hashiyada M, Sato K, Nata M, Funato T, Okamura K.: "Complete XY gonadal dysgenesis and aspects of the SRY genotype and gonadal tumor formation"J Hum Genet. 47. 279-284 (2002)

Uehara S、Hashiyada M、Sato K、Nata M、Funato T、Okamura K.：“完全 XY 性腺发育不全以及 SRY 基因型和性腺肿瘤形成的方面”J Hum Genet。

Sato K, Hashiyada M, Uehara S, Nata M, Okamura K: "CpG dinucleotide methylation patterns in the human androgen receptor gene and X-chromosome inactivation in peripheral blood leukocytes of phenotypically normal women"J Hum Genet. 48. 374-379 (2003)

Sato K、Hashiyada M、Uehara S、Nata M、Okamura K：“人雄激素受体基因中的 CpG 二核苷酸甲基化模式和表型正常女性外周血白细胞中的 X 染色体失活”J Hum Genet。

那谷雅之, 橋谷田真樹, 舟山眞人, 安達 登: "航空機墜落現場から発見された多数骨片の個人識別"DNA多型. 10. 274-278 (2002)

Masayuki Natani、Maki Hashiyada、Masato Funayama、Noboru Adachi：“飞机失事现场发现的大量骨头碎片的个人鉴定”DNA 多态性。