Single nucleotide polymorphism genotyping by on-line liquid chromatography-mass spectrometry in forensic science of the Y-chromosomal locus M9
Single nucleotide polymorphism genotyping by on-line liquid chromatography-mass spectrometry in forensic science of the Y-chromosomal locus M9
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DOI：
10.1016/s1570-0232(02)00694-3
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发表时间：
2002-12-25
影响因子：
3
通讯作者：
Parson, W
中科院分区：
文献类型：
--
作者：
Berger, B;Hölzl, G;Parson, W
描述了一种用于 Y 染色体位点 M9 等位基因基因分型的方法，包括 DNA 提取、聚合酶链式反应 (PCR) 扩增、离子对反相高效液相色谱 (IP-RP-HPLC) 样品纯化以及在线连字电喷雾电离质谱 (ESI-MS) 等位基因鉴定。基于完整的分子质量测量，等位基因 G 和 C 在 114 个碱基对扩增子中进行区分，质量精度在 0.007% 至 0.017% 之间。扩增 61 bp 短片段后，质量测定的准确度显着降低至低于 0.0036%。在 25 mM 丁基二甲基碳酸氢铵中应用乙腈的陡梯度不仅能够在基于聚（苯乙烯-二乙烯基苯）的整体毛细管柱中从 PCR 产物中有效分离非目标成分，而且还允许在 2 分钟的循环时间内对 PCR 产物进行高通量分析。将新方法与毛细管凝胶电泳分析的传统限制性片段长度多态性测定进行比较。在一项盲法研究中，对 90 个无关个体的样本进行了基因分型。高精度（
A method is described for genotyping alleles of the Y-chromosomal locus M9, incorporating DNA extraction, amplification by polymerase chain reaction (PCR), sample purification by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC), and allele identification by on-line hyphenation to electrospray ionization mass spectrometry (ESI-MS). The alleles G and C were differentiated in 114 base pair amplicons on the basis of intact molecular mass measurements with a mass accuracy between 0.007 and 0.017%. The accuracy of mass determination was significantly reduced to less than 0.0036% upon amplification of a short, 61 bp fragment. The application of steep gradients of acetonitrile in 25 mM butyldimethylammonium bicarbonate not only enabled the efficient separation of non-target components from the PCR product in a monolithic, poly-(styrene-divinylbenzene)-based capillary column, but also allowed the high-throughput analysis of the PCR products with cycle times of 2 min. The new method was compared to a conventional restriction fragment length polymorphism assay with capillary gel electrophoretic analysis. In a blind study, 90 samples of unrelated individuals were genotyped. The high accuracy (