AUTOMATIC DETECTION SYSTEM OF GENETIC POLYMORPHISMS

遗传多态性自动检测系统

• 批准号: 10557074
• 负责人: NARISAWA Kuniaki
• 金额: $ 5.76万
• 依托单位: TOHOKU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10557074/
• 关键词: KNOWN MUTATION; TAQ MAN-ASA; SYBR-GREEN-ASA; ALLELE SPECIFIC PRIMER; PHENYLKETONURIA; GLYCOGEN STORAGE DISEASE TYPE 1A; MEDIUM CHAIN ACYL-COA DEHYDROGENASE; Multiplex primer extension法; Extention プライマー; 糖原病1型; ホロカルボキシラーゼ合成酵素欠損症; Tay-Sachs病; 軟骨

We have developed two novel methods for the detection of point mutations, TaqMan-ASA and SYBR Green-ASA. APCR amplicon using two sets of allele-specific primers in the presence of a TaqMan probe or SYBR Green dye was monitored in real time with a fluorescence detector (PRISM 7700 Sequence Detection System). The difference in amplification efficiency between the two PCR reactions was determined by "threshold" cycles to differentiate mutant and normal alleles. These methods eliminate the requirement for subsequent gel electrophoresis or additional hybridization steps by directly detecting positive reactions. We applied these methods to detect R111X, IVS-4, Y204C, R241C, R243Q, R245V, R252W, R278W, IVS-9, Y356X and R413P mutations in patients with phenylketonuria, a prevalent 727g>t mutation in Japanese patients with glycogen storage disease type la, and a common K329E mutation in Caucasian patients with medium-chain acyl-CoA dehydrogenase deficiency. These techniques can be automated and are useful for the genotype analysis of a variety of single nucleotide polymorphisms and mutations.

我们开发了两种检测点突变的新方法：TaqMan-ASA 和 SYBR Green-ASA。使用荧光检测器（PRISM 7700 序列检测系统）实时监测在 TaqMan 探针或 SYBR Green 染料存在下使用两组等位基因特异性引物的 APCR 扩增子。通过“阈值”循环来确定两个 PCR 反应之间扩增效率的差异，以区分突变体和正常等位基因。这些方法通过直接检测阳性反应，消除了后续凝胶电泳或额外杂交步骤的需要。我们应用这些方法检测苯丙酮尿症患者中的 R111X、IVS-4、Y204C、R241C、R243Q、R245V、R252W、R278W、IVS-9、Y356X 和 R413P 突变、日本 la 型糖原贮积病患者中普遍存在的 727g>t 突变以及常见的 K329E 中链酰基辅酶A脱氢酶缺乏症白种人患者的突变。这些技术可以自动化，可用于多种单核苷酸多态性和突变的基因型分析。

项目成果

Sakamoto O. et al.: "Relationship between kinetic properties of mutant enzymes and biochemaical and clinical responsiveness to biotin in holocarboxylase synthetase deficiency"Pediatric Research. 46,6. 671-676 (1999)

Sakamoto O. 等人：“全羧化酶合成酶缺乏症中突变酶的动力学特性与生物化学和临床对生物素的反应之间的关系”儿科研究。

Fujii K.et al.: "Mutation detection by TaqMan-allele specific amplification : Application to molecular diagnosis of glycogen storage disease type Ia and medium-chain acyl-CoA dehydrogenase deficiency"Human Mutation. 15,2. 189-196 (2000)

Fujii K.等人：“通过 TaqMan 等位基因特异性扩增进行突变检测：应用于 Ia 型糖原贮积病和中链酰基辅酶 A 脱氢酶缺乏症的分子诊断”人类突变。

Hou D.et al.: "Glycogen storage disease type Ib : structural and mutation analysis of the microsomal glucose-6-phosphate transporter gene"American Journal of Human Genetics. 86,3. 254-257 (1999)

侯D.等人：“Ib型糖原贮积病：微粒体葡萄糖-6-磷酸转运蛋白基因的结构和突变分析”美国人类遗传学杂志。

Sakamoto, O, et al.: "Diagnosis and mutation analysis of an atypical case of holocarboxylase synthetase deficiency"Europian Journal of Pediatrics. 159,1-2. 18-22 (2000)

Sakamoto, O, et al.：“全羧化酶合成酶缺乏症非典型病例的诊断和突变分析”欧洲儿科杂志。

Aoki, Y, et al.: "Identification and characterization of mutations in patients with holocarboxylase synthetase deficiency"Human Genetics. 104. 143-148 (1999)

Aoki, Y 等人：“全羧化酶合成酶缺乏症患者突变的鉴定和表征”人类遗传学。