Mutation detection by TaqMan‐allele specific amplification: Application to molecular diagnosis of glycogen storage disease type Ia and medium‐chain acyl‐CoA dehydrogenase deficiency
Mutation detection by TaqMan‐allele specific amplification: Application to molecular diagnosis of glycogen storage disease type Ia and medium‐chain acyl‐CoA dehydrogenase deficiency
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DOI：
10.1002/(sici)1098-1004(200002)15:2
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发表时间：
2000-02
期刊：
影响因子：
3.9
通讯作者：
Kunihiro Fujii;Y. Matsubara;J. Akanuma;Kazutoshi Takahashi;S. Kure;Yoichi Suzuki;M. Imaizumi;K. Iinuma;O. Sakatsume;P. Rinaldo;K. Narisawa
中科院分区：
文献类型：
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作者：
Kunihiro Fujii;Y. Matsubara;J. Akanuma;Kazutoshi Takahashi;S. Kure;Yoichi Suzuki;M. Imaizumi;K. Iinuma;O. Sakatsume;P. Rinaldo;K. Narisawa
我们设计了一种使用TaqMan荧光探针（阿萨）的等位基因特异性扩增方法，用于检测点突变。在TaqMan探针存在下使用两组等位基因特异性引物进行的成对PCR扩增用荧光检测器进行真实的实时监测。通过“阈值”循环确定两个PCR反应之间的扩增效率差异，以在没有PCR后处理的情况下区分突变和正常等位基因。该方法测量的是扩增效率，而不是终点PCR产物的存在与否，因此在设计等位基因特异性引物时具有更大的灵活性，并为等位基因区分提供了充足的技术裕度。我们应用阿萨方法检测了日本Ia型糖原累积病患者中常见的727 G>T突变和高加索中链酰基辅酶A脱氢酶缺乏症患者中常见的985 A>G突变。该方法可实现自动化，并可应用于各种遗传性疾病的DNA诊断。2000年，《Mutat》第15卷，第189-196页。© 2000 Wiley利斯公司
We have devised an allele‐specific amplification method with a TaqMan fluorogenic probe (TaqMan‐ASA) for the detection of point mutations. Pairwise PCR amplification using two sets of allele‐specific primers in the presence of a TaqMan probe was monitored in real time with a fluorescence detector. Difference in amplification efficiency between the two PCR reactions was determined by “threshold” cycles to differentiate mutant and normal alleles without post‐PCR processing. The method measured the efficiency of amplification rather than the presence or absence of end‐point PCR products, therefore allowing greater flexibility in designing allele‐specific primers and an ample technical margin for allelic discrimination. We applied the TaqMan‐ASA method to detect a prevalent 727G>T mutation in Japanese patients with glycogen storage disease type Ia and a common 985A>G mutation in Caucasian patients with medium‐chain acyl‐CoA dehydrogenase deficiency. The method can be automated and may be applicable to the DNA diagnosis of various genetic diseases. Hum Mutat 15:189–196, 2000. © 2000 Wiley‐Liss, Inc.