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首页> 外文期刊>Investigative Genetics >Development of a fast PCR protocol enabling rapid generation of AmpF?STR? Identifiler? profiles for genotyping of human DNA
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Development of a fast PCR protocol enabling rapid generation of AmpF?STR? Identifiler? profiles for genotyping of human DNA

机译：快速PCR方案的开发，可快速生成AmpF？STR？身份识别器？人类DNA基因分型的概况

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Background Traditional PCR methods for forensic STR genotyping require approximately 2.5 to 4 hours to complete, contributing a significant portion of the time required to process forensic DNA samples. The purpose of this study was to develop and validate a fast PCR protocol that enabled amplification of the 16 loci targeted by the AmpF?STR? Identifiler? primer set, allowing decreased cycling times. Methods Fast PCR conditions were achieved by substituting the traditional Taq polymerase for SpeedSTAR? HS DNA polymerase which is designed for fast PCR, by upgrading to a thermal cycler with faster temperature ramping rates and by modifying cycling parameters (less time at each temperature) and adopting a two-step PCR approach. Results The total time required for the optimized protocol is 26 min. A total of 147 forensically relevant DNA samples were amplified using the fast PCR protocol for Identifiler. Heterozygote peak height ratios were not affected by fast PCR conditions, and full profiles were generated for single-source DNA amounts between 0.125 ng and 2.0 ng. Individual loci in profiles produced with the fast PCR protocol exhibited average n-4 stutter percentages ranging from 2.5 ± 0.9% (THO1) to 9.9 ± 2.7% (D2S1338). No increase in non-adenylation or other amplification artefacts was observed. Minor contributor alleles in two-person DNA mixtures were reliably discerned. Low level cross-reactivity (monomorphic peaks) was observed with some domestic animal DNA. Conclusions The fast PCR protocol presented offers a feasible alternative to current amplification methods and could aid in reducing the overall time in STR profile production or could be incorporated into a fast STR genotyping procedure for time-sensitive situations.
机译：背景技术用于法医STR基因分型的传统PCR方法大约需要2.5到4个小时才能完成，这占了法医DNA样品处理时间的很大一部分。这项研究的目的是开发和验证一种快速PCR方案，该方案能够扩增AmpF？STR ？靶向的16个基因座。 标识符？ 引物组，可以减少循环时间。方法用传统的Taq聚合酶代替SpeedSTAR实现快速PCR条件。 HS DNA聚合酶设计用于快速PCR，方法是升级到具有更快温度升高速率的热循环仪，并修改循环参数（每个温度下花费的时间更少），并采用两步PCR方法。结果优化方案所需的总时间为26分钟。使用Identifiler的快速PCR方案扩增了总共147个法医相关的DNA样品。杂合子的峰高比不受快速PCR条件的影响，对于单源DNA量介于0.125 ng和2.0 ng之间的分子，可生成完整的图谱。用快速PCR方案产生的谱图中的单个基因座显示出平均n-4口吃百分比，范围从2.5±0.9％（THO1）到9.9±2.7％（D2S1338）。没有观察到非腺苷酸化或其他扩增伪像的增加。可靠地识别了两人DNA混合物中的次要贡献等位基因。与一些家畜DNA观察到低水平的交叉反应性（单峰）。结论提出的快速PCR方案为当前扩增方法提供了一种可行的替代方法，可以帮助减少STR谱图生成的总时间，或者可以将其纳入对时间敏感的情况的快速STR基因分型程序中。

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