A multiplex allele-specific primer extension assay for forensically informative SNPs distributed throughout the mitochondrial genome
A multiplex allele-specific primer extension assay for forensically informative SNPs distributed throughout the mitochondrial genome
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DOI：
10.1007/s00414-004-0428-5
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发表时间：
2004-06-01
影响因子：
2.1
通讯作者：
Parsons, TJ
中科院分区：
文献类型：
--
作者：
Vallone, PM;Just, RS;Parsons, TJ
位于整个线粒体基因组（mtGenome）的单核苷酸多态性（SNP）的分型可以帮助解决具有相同HV 1/HV 2线粒体型的个体。一组11个SNPs的选择区分最常见的高加索人HV 1/HV 2有丝分裂型的个人被纳入等位基因特异性引物延伸试验。优化该测定以用于多重检测mtGenome中位置3010、4793、10211、5004、7028、7202、16519、12858、4580、477和14470处的SNP。设计引物以允许同时PCR扩增mtGenome中的11个独特区域和随后的引物延伸。通过将荧光标记的双脱氧核苷酸（ddNTPs）酶促掺入到延伸引物的3'末端上，可以使用大多数法医实验室中常见的基于毛细管的电泳（CE）平台来完成检测。在变性POP 4和POP 6 CE运行缓冲液中比较延伸引物的电泳迁移率。延伸引物浓度的经验调整导致探测的11个基因座的均匀信号强度。我们证明，该检测方法表现良好的异质性和混合物检测，并为典型的mtDNA个案样本与高度降解的DNA。
The typing of single nucleotide polymorphisms (SNPs) located throughout the mitochondrial genome (mtGenome) can help resolve individuals with an identical HV1/HV2 mitotype. A set of 11 SNPs selected for distinguishing individuals of the most common Caucasian HV1/HV2 mitotype were incorporated in an allele specific primer extension assay. The assay was optimized for multiplex detection of SNPs at positions 3010, 4793, 10211, 5004, 7028, 7202, 16519, 12858, 4580, 477 and 14470 in the mtGenome. Primers were designed to allow for simultaneous PCR amplification of 11 unique regions in the mtGenome and subsequent primer extension. By enzymatically incorporating fluorescently labeled dideoxynucleotides (ddNTPs) onto the 3' end of the extension primer, detection can be accomplished with a capillary-based electrophoresis (CE) platform common in most forensic laboratories. The electrophoretic mobility for the extension primers was compared in denaturing POP4 and POP6 CE running buffers. Empirical adjustment of extension primer concentrations resulted in even signal intensity for the 11 loci probed. We demonstrate that the assay performs well for heteroplasmy and mixture detection, and for typical mtDNA casework samples with highly degraded DNA.