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首页> 外文期刊>BMC Medical Genomics >Robust SNP genotyping by multiplex PCR and arrayed primer extension
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Robust SNP genotyping by multiplex PCR and arrayed primer extension

机译：通过多重PCR和阵列引物延伸进行可靠的SNP基因分型

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Background Arrayed primer extension (APEX) is a microarray-based rapid minisequencing methodology that may have utility in 'personalized medicine' applications that involve genetic diagnostics of single nucleotide polymorphisms (SNPs). However, to date there have been few reports that objectively evaluate the assay completion rate, call rate and accuracy of APEX. We have further developed robust assay design, chemistry and analysis methodologies, and have sought to determine how effective APEX is in comparison to leading 'gold-standard' genotyping platforms. Our methods have been tested against industry-leading technologies in two blinded experiments based on Coriell DNA samples and SNP genotype data from the International HapMap Project. Results In the first experiment, we genotyped 50 SNPs across the entire 270 HapMap Coriell DNA sample set. For each Coriell sample, DNA template was amplified in a total of 7 multiplex PCRs prior to genotyping. We obtained good results for 41 of the SNPs, with 99.8% genotype concordance with HapMap data, at an automated call rate of 94.9% (not including the 9 failed SNPs). In the second experiment, involving modifications to the initial DNA amplification so that a single 50-plex PCR could be achieved, genotyping of the same 50 SNPs across each of 49 randomly chosen Coriell DNA samples allowed extremely robust 50-plex genotyping from as little as 5 ng of DNA, with 100% assay completion rate, 100% call rate and >99.9% accuracy. Conclusion We have shown our methods to be effective for robust multiplex SNP genotyping using APEX, with 100% call rate and >99.9% accuracy. We believe that such methodology may be useful in future point-of-care clinical diagnostic applications where accuracy and call rate are both paramount.
机译：背景技术阵列引物延伸（APEX）是一种基于微阵列的快速微测序方法，可用于涉及单个核苷酸多态性（SNP）遗传诊断的“个性化医学”应用。但是，迄今为止，很少有报告客观地评估化验完成率，调用率和APEX的准确性。我们进一步开发了可靠的测定设计，化学和分析方法，并试图确定与领先的“金标准”基因分型平台相比APEX的有效性。我们的方法已经在两项基于Coriell DNA样本和来自国际HapMap项目的SNP基因型数据的盲法实验中，针对行业领先的技术进行了测试。结果在第一个实验中，我们对整个270个HapMap Coriell DNA样本集中的50个SNP进行了基因分型。对于每个Coriell样品，在进行基因分型之前，应在总共7个多重PCR中扩增DNA模板。我们获得了41个SNP的良好结果，其基因型与HapMap数据的符合率为99.8％，自动调用率为94.9％（不包括9个失败的SNP）。在第二个实验中，涉及到对初始DNA扩增的修饰，从而可以实现单个50重PCR，对49个随机选择的Coriell DNA样品中的每个相同的50个SNP进行基因分型，从最少的50个基因就可以进行极其鲁棒的50重基因分型。 5 ng DNA，分析完成率100％，调用率100％，准确度> 99.9％。结论我们已经证明了我们的方法对于使用APEX进行鲁棒的多重SNP基因分型是有效的，呼叫率100％，准确度> 99.9％。我们相信，这种方法可能在未来的即时医疗点临床诊断应用中很有用，因为准确性和呼叫率都是至关重要的。

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