一种在单一聚合酶链反应检测中使用碱基淬灭探针技术检测线粒体C1494T和A1555G突变的新方法
[A novel method of detecting mitochondrial C1494T and A1555G mutations by using the base-quenched probe technique in a single PCR assay].
作者信息
ZHENG Lu, LUO Guang-hua, ZHANG Jun, ZHANG Xiao-ying, XU Ning
机构信息
Comprehensive Laboratoty, Third Affiliated Hospital, Soochow University, Changzhou 213003, China.
出版信息
Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi. 2012 Apr;47(4):326-9.
OBJECTIVE
In the present study we describe a new method to detect the mitochondrial DNA (mtDNA) mutations A1555G and C1494T by using the base-quenched probe technique in a single PCR reaction.
METHODS
6-carboxyfluorescein (FAM) was directly conjugated to the 3' end of the probe. Four vectors, representing the four possible genotype combinations, were constructed as the amplification template for the methodology established. In present study A1555G and C1494T mutations in 117 individuals with hearing loss were detected by the base-quenched probe method and were further validated by the direct DNA sequencing analyses.
RESULTS
From the melting curve we could distinguish the four haplotypes accurately. And there were complete concordance between the base-quenched probe method and direct DNA sequencing.
CONCLUSION
This method is suitable for clinical test of mtDNA mutations A1555G and C1494T in individuals with hearing loss.
目的
在本研究中，我们描述了一种通过在单个聚合酶链反应（PCR）中使用碱基淬灭探针技术来检测线粒体DNA（mtDNA）突变A1555G和C1494T的新方法。
方法
6-羧基荧光素（FAM）直接与探针的3'末端偶联。构建了代表四种可能基因型组合的四个载体，作为所建立方法的扩增模板。在本研究中，通过碱基淬灭探针法检测了117例听力损失患者中的A1555G和C1494T突变，并通过直接DNA测序分析进一步验证。
结果
从熔解曲线我们可以准确区分四种单倍型。并且碱基淬灭探针法与直接DNA测序之间完全一致。
结论
该方法适用于听力损失患者mtDNA突变A1555G和C1494T的临床检测。
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