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.

我们开发了两种新的方法来检测点突变，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APCR扩增子在Taqman探针或SYBR绿色染料存在下使用两组等位基因特异性引物。通过“阈值”循环确定了两个PCR反应之间的扩增效率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来消除对随后的凝胶电泳或其他杂交步骤的要求。我们将这些方法应用于检测R111X，IVS-4，Y204C，R241C，R243Q，R245V，R252W，R252W，R278W，R278W，IVS-9，Y356X和R413P突变患者患有苯基尿布病患者，患有727G> T的患者，日本患者患有GGY> T突变。 LA型和中链酰基-COA脱氢酶缺乏症的白种人患者中的常见K329E突变。这些技术可以是自动化的，对于多种单核苷酸多态性和突变的基因型分析非常有用。

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。

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。

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。

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。