Zhang S, Van Pelt C K, Schultz G A
Advion BioSciences, Inc., Ithaca, New York 14850, USA.
Anal Chem. 2001 May 1;73(9):2117-25. doi: 10.1021/ac001549j.
The high frequency of single nucleotide polymorphisms (SNPs) in the human genome makes them ideal genetic markers for mapping, diagnosing disease-related alleles, and identifying SNPs that contribute to drug response differences between individuals. Here we report a novel assay utilizing a single nucleotide primer extension (SNuPE) and electrospray ionization mass spectrometry (ESI-MS) detection for the analysis of SNPs. In contrast to most SNuPE genotyping technologies that detect the extended primer product, the novel Survivor assay detects the unreacted dideoxynucleotides (ddNTPs) remaining or surviving in solution following a SNuPE. This assay involves a simple analysis of the same four ddNTP analytes, regardless of the SNP being investigated, and either single or double-stranded DNA can be used to genotype a SNP, without any labeling requirements of the ddNTPs or oligonucleotide primers. We have tested and blindly validated the Survivor assay by genotyping the C/T SNP at -857 of the human TNFalpha promoter gene. The results obtained are in agreement with the control sequencing data. The results demonstrate that the homogeneous Survivor assay with ESI-MS detection offers advantages in simplicity, accuracy, specificity, and sensitivity. Additional advantages of the method include enhanced hybridization efficiencies in this solution-phase assay and the elimination of immobilized primers for the isolation of single-stranded DNA. With a one-well reaction and an automation platform being developed, the Survivor assay provides a powerful new tool for large-scale SNP analysis and screening.
人类基因组中单个核苷酸多态性（SNP）的高频率使其成为用于图谱绘制、诊断疾病相关等位基因以及识别导致个体间药物反应差异的SNP的理想遗传标记。在此，我们报告一种利用单核苷酸引物延伸（SNuPE）和电喷雾电离质谱（ESI-MS）检测来分析SNP的新型检测方法。与大多数检测延伸引物产物的SNuPE基因分型技术不同，新型Survivor检测方法检测的是在SNuPE之后残留在溶液中或“存活”下来的未反应双脱氧核苷酸（ddNTP）。该检测方法对相同的四种ddNTP分析物进行简单分析，而无需考虑所研究的SNP，并且单链或双链DNA均可用于SNP基因分型，无需对ddNTP或寡核苷酸引物进行任何标记。我们通过对人类TNFalpha启动子基因 -857位的C/T SNP进行基因分型，对Survivor检测方法进行了测试和盲法验证。所得结果与对照测序数据一致。结果表明，采用ESI-MS检测的均相Survivor检测方法在简便性、准确性、特异性和灵敏度方面具有优势。该方法的其他优点包括在这种溶液相检测中提高了杂交效率，以及无需固定引物来分离单链DNA。随着单孔反应和正在开发的自动化平台的出现，Survivor检测方法为大规模SNP分析和筛选提供了一种强大的新工具。
