RAPID AND ACCURATE GENOTYPING OF STRS BY ESI-MS

这项研究的目的是通过开发一种基于电喷雾电离质谱仪的快速准确的单核苷酸多态(SNPs)和短串联重复序列(STR)基因分型的新技术，为理解和诊断人类遗传性疾病做出贡献。聚合酶链式反应与质谱仪联用，由于其快速、准确和精确的质量测量，以及通过气相测序推断一级结构的固有能力，是一种近乎理想的基因分型平台。拟议的工作最初将侧重于阐明制备专门用于电喷雾电离质谱仪的聚合酶链式反应产物的最佳策略，这是一项艰巨的挑战。沉淀法或基于生物亲和力捕获的更精细的方法；这两种策略都可以在96孔格式中自动进行。一旦在拟议研究的最初12-15个月内完成了快速准备战略，验证研究将启动无凝胶、无芯片的技术，这种技术对于SNPs和STR的基因分型是快速、准确和可靠的，可以实际放置在临床环境中。验证研究完成后，将从90个SNP小组中对未知基因座进行基因分型，以确定STR和SNP图谱之间是否存在任何相关性，首先是位于7号染色体上的T细胞受体β基因座。同时，将对导致人类耳聋的基因进行基因分型。所有的基因类型都将通过数据库的开发迅速传播给公众，该数据库包括与特定多态基因座有关的其他相关信息的超级链接。

The objective of the proposed research are to contribute to the understanding and diagnosis of genetic human diseases by the development of a new technology based upon electrospray ionization mass spectrometry for rapid and accurate genotyping of single-nucleotide polymorphisms (SNPs) and short tandem repeats (STRs). Polymerase chain reaction (PCR) coupled with mass spectrometry is a near ideal platform for genotyping due to its high speed, accurate and precise mass measurements, and having the inherent ability to deduce primary structure via gas-phase sequencing. The proposed work will initially focus on elucidating the best strategy to prepare PCR products specifically for electrospray ionization mass spectrometry which is a formidable challenge. The precipitation or a more elaborate approach based on bio- affinity capture; both strategies can be automated in a 96-well format. Once a rapid preparation strategy is accomplished in the first 12-15 months of the proposed research, validation studies will be initiated gel- less, chip-less technology that is rapid, accurate and reliable for genotyping of SNPs and STRs which could realistically be placed in a clinical setting. Upon completion of the validation studies, unknown loci will be genotyped form the SNP panel of 90 to determine if there are any correlations between STR and SNP maps starting with the T-cell receptor beta loci located on chromosome 7. Concurrently, genes responsible for human deafness will be genotyped. All genotypes will be rapidly disseminated to the public through the development of a database including hyperlinks to other pertinent information regarding a specific polymorphic locus.