高通量飞行时间质谱基因分型方法的研究

为了考察飞行时间质谱基因分型方法 (MALDI-TOF) 的位点分型成功率和分型结果质量的关系，分析了 96 个 SNPs 位点的近 10 000 个基因分型数据 (用 MALDI-TOF “4 重”实验方法检测 ). 结果显示，位点分型成功率和分型结果的质量显著正相关 . 分型成功率低于 82% 的 SNP 位点，其高质量结果占的比例开始逐渐降低 . 提示 82% 的分型成功率可以作为衡量分型结果质量的数据点 . 为了进一步提高通量并降低成本，在 MALDI-TOF “ 4 重”实验方法的基础上，发展了两种“准 8 重”实验方法 . 用新的实验方法检测了 95 个样本的 32 个 SNPs 位点 . 结果显示“混合准 8 重”实验方法与“ 4 重”实验方法相比无显著差异，而“复点准 8 重”的结果差于“ 4 重”分型方法 .

The Study of High Throughput MALDI-TOF Genotyping Assay

MALDI-TOF is a simple and robust method for the analysis of single nucleotide polymorphism (SNP), which combines proven high-fidelity enzymatic procedures and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry. This technology is among the most promising SNP typing methods for delivering accurate, effective, flexible and high throughput analysis of SNPs. "4-plex" genotyping protocol, which could detect 4 SNPs in one reaction, was performed in Beijing Genomics Institute with accuracy of 99.55%. To learn the correlation between genotyping call rates and quality, nearly ten thousands of typing results ("4-plex") were analyzed. Significant positive correlation between call rate and the counts of conservative results were observed. SNPs with call rate lower than 82% have less conservative results, suggesting 82% was a cutoff to evaluate the genotyping results of MALDI-TOF assay. Aiming to further improve genotyping throughput and reduce the cost, "Mix 8-plex" and "Double-spotting 8-plex" genotyping protocols were developed. For new protocols, PCR and primer extension reactions were still performed under reliable "4-plex" protocol. For "Mix 8-plex" protocol, two sets of "4-plex" primer extension products were mixed and dispensed onto one SpectroCHIP at the same time. As to "Double-spotting 8-plex" protocol, two sets of "4-plex" products were consecutively dispensed onto one SpectroCHIP. 32 SNPs were genotyped in 95 human DNA samples to test the feasibility of these new protocols. The results showed that the performance of "Mix 8-plex" was as good as "4-plex" protocol, while the performance of "Double-spotting 8-plex" was poor.