Hopgood R, Sullivan K M, Gill P
Central Research and Support Establishment, Home Office Forensic Science Service, Reading, U.K.
Biotechniques. 1992 Jul;13(1):82-92.
A rapid, robust and sensitive method has been developed for the amplification and direct sequencing of human mitochondrial DNA. A 403-bp hypervariable segment was amplified by two successive rounds of nested PCR. This was then sequenced by the dideoxy chain termination method using dye-labeled universal sequencing primers in conjunction with an automated DNA sequencer. This paper describes the assessment of four different strategies for this amplification and sequencing process. Optimal results were obtained by immobilizing the biotinylated PCR product on Dynabeads followed by solid-phase sequencing with Sequenase. Degraded samples and those containing trace amounts of DNA such as extracts from hair shafts can be analyzed by this method.
已开发出一种快速、稳健且灵敏的方法用于人类线粒体DNA的扩增和直接测序。通过两轮连续的巢式PCR扩增出一段403bp的高变区。然后使用染料标记的通用测序引物结合自动DNA测序仪，通过双脱氧链终止法对其进行测序。本文描述了对该扩增和测序过程的四种不同策略的评估。通过将生物素化的PCR产物固定在磁珠上，然后用Sequenase进行固相测序，可获得最佳结果。该方法可分析降解的样本以及含有微量DNA的样本，如毛发轴提取物。
