Alderborn A, Kristofferson A, Hammerling U
Research & Development, Pyrosequencing AB, Uppsala, Sweden.
Genome Res. 2000 Aug;10(8):1249-58. doi: 10.1101/gr.10.8.1249.
The characterization of naturally occurring variations in the human genome has evoked an immense interest during recent years. Variations known as biallelic Single-Nucleotide Polymorphisms (SNPs) have become increasingly popular markers in molecular genetics because of their wide application both in evolutionary relationship studies and in the identification of susceptibility to common diseases. We have addressed the issue of SNP genotype determination by investigating variations within the Renin-Angiotensin-Aldosterone System (RAAS) using pyrosequencing, a real-time pyrophosphate detection technology. The method is based on indirect luminometric quantification of the pyrophosphate that is released as a result of nucleotide incorporation onto an amplified template. The technical platform employed comprises a highly automated sequencing instrument that allows the analysis of 96 samples within 10 to 20 minutes. In addition to each studied polymorphic position, 5-10 downstream bases were sequenced for acquisition of reference signals. Evaluation of pyrogram data was accomplished by comparison of peak heights, which are proportional to the number of incorporated nucleotides. Analysis of the pyrograms that resulted from alternate allelic configurations for each addressed SNP revealed a highly discriminating pattern. Homozygous samples produced clear-cut single base peaks in the expected position, whereas heterozygous counterparts were characterized by distinct half-height peaks representing both allelic positions. Whenever any of the allelic bases of an SNP formed a homopolymer with adjacent bases, the nonallelic signal was added to those of the SNP. This feature did not, however, influence SNP readability. Furthermore, the multibase reading capacity of the described system provides extensive flexibility in regard to the positioning of sequencing primers and allows the determination of several closely located SNPs in a single run.
近年来，对人类基因组中自然发生变异的特征描述引起了极大的关注。被称为双等位基因单核苷酸多态性（SNP）的变异，因其在进化关系研究和常见疾病易感性鉴定中的广泛应用，已成为分子遗传学中越来越受欢迎的标记。我们通过使用焦磷酸测序法（一种实时焦磷酸检测技术）研究肾素 - 血管紧张素 - 醛固酮系统（RAAS）内的变异，来解决SNP基因型测定的问题。该方法基于对由于核苷酸掺入扩增模板而释放的焦磷酸的间接发光定量。所采用的技术平台包括一台高度自动化的测序仪，可在10至20分钟内分析96个样本。除了每个研究的多态性位点外，还对5 - 10个下游碱基进行测序以获取参考信号。通过比较与掺入核苷酸数量成比例的峰高来完成对焦磷酸测序图数据的评估。对每个所研究SNP的交替等位基因构型产生的焦磷酸测序图的分析揭示了一种高度区分性的模式。纯合样本在预期位置产生清晰的单碱基峰，而异合样本则以代表两个等位基因位置的明显半高峰为特征。每当SNP的任何一个等位基因碱基与相邻碱基形成同聚物时，非等位基因信号就会加到SNP的信号上。然而，这一特征并不影响SNP的可读性。此外，所述系统的多碱基读取能力在测序引物的定位方面提供了广泛的灵活性，并允许在一次运行中测定几个紧密相邻的SNP。
