Wang Jun, Chuang Karen, Ahluwalia Mandeep, Patel Sarika, Umblas Nanette, Mirel Daniel, Higuchi Russell, Germer Soren
Human Genetics Department, Roche Molecular Systems, Alameda, CA 94501, USA.
Biotechniques. 2005 Dec;39(6):885-93. doi: 10.2144/000112028.
Despite many recent advances in high-throughput single nucleotide polymorphism (SNP) genotyping technologies, there is still a great need for inexpensive and flexible methods with a reasonable throughput. Here we report substantial modifications and improvements to an existing homogenous allele-specific PCR-based SNP genotyping method, making it an attractive new option for researchers engaging in candidate gene studies or following up on genome-wide scans. In this advanced version of the melting temperature (Tm)-shift SNP genotyping method, we attach two GC-rich tails of different lengths to allele-specific PCR primers, such that SNP alleles in genomic DNA samples can be discriminated by the Tms of the PCR products. We have validated 306 SNP assays using this method and achieved a success rate in assay development of greater than 83% under uniform PCR conditions. We have developed a standalone software application to automatically assign genotypes directly from melting curve data. To demonstrate the accuracy of this method, we typed 592 individuals for 6 SNPs and showed a high call rate (>98%) and high accuracy (>99.9%). With this method, 6-10,000 samples can be genotyped per day using a single 384-well real-time thermal cycler with 2-4 standard 384-well PCR instruments.
尽管高通量单核苷酸多态性（SNP）基因分型技术最近取得了许多进展，但对于具有合理通量的廉价且灵活的方法仍有很大需求。在此，我们报告了对现有的基于等位基因特异性PCR的SNP基因分型方法的重大改进，使其成为从事候选基因研究或跟进全基因组扫描的研究人员的一个有吸引力的新选择。在这个熔解温度（Tm）位移SNP基因分型方法的先进版本中，我们将两条不同长度的富含GC的尾巴连接到等位基因特异性PCR引物上，这样基因组DNA样本中的SNP等位基因就可以通过PCR产物的Tm值来区分。我们使用这种方法验证了306个SNP检测，并且在统一的PCR条件下检测开发的成功率超过83%。我们开发了一个独立的软件应用程序，可直接从熔解曲线数据自动确定基因型。为了证明这种方法的准确性，我们对592个个体进行了6个SNP的分型，显示出高检出率（>98%）和高精度（>99.9%）。使用这种方法，每天使用一台384孔实时热循环仪和2 - 4台标准384孔PCR仪器可以对6000 - 10000个样本进行基因分型。
