CAREER: Nucleic Acid Mutation Detection by 2'-Amine Modification

在这项研究中，PI将使用一种新的化学方法来检测核酸序列中的点突变和其他缺陷。该方法使用其中一个2‘-核糖位置(分别在DNA或RNA中的-H或-OH)被胺(-NH2)取代的寡核苷酸探针。2‘-胺取代的核苷酸被琥珀酰亚胺基酯特异性地酰化，形成2’-酰胺产物。该突变检测方法利用了PI出人意料的观察，即错配位置的2‘-胺基团的酰化速度比碱基配对核苷酸上的胺取代更快。由于增加了局部灵活性，酰化专用于不匹配的2‘-胺基团。2‘-胺取代的探针寡核苷酸和靶序列都可以是DNA或RNA。该方法在查询错配和定量核酸群体中具有单碱基特异性的杂交方面具有广泛的潜在用途。此外，活化剂是一种双功能分子，其中突变敏感的酰化化学连接到一个独立的可视化部分。这个职业奖的目标是，首先，从根本上探索这种化学传感器技术的应用、机理和局限性，然后，开发一种实用的方法来可视化人类基因的突变。国际生物化学研究所为高级生化实验室提出了新的实验，包括基于研究的实验和生物信息学模块，并建立了一门基于文献的酶机制课程。9984289周在这项建议中，国际生物化学研究所将使用一种新的化学方法来检测核酸序列中的点突变和其他缺陷。该方法使用其中一个2‘-核糖位置(分别在DNA或RNA中的-H或-OH)被胺(-NH2)取代的寡核苷酸探针。2‘-胺取代的核苷酸被琥珀酰亚胺基酯特异性地酰化，形成2’-酰胺产物。该突变检测方法利用了PI出人意料的观察，即错配位置的2‘-胺基团的酰化速度比碱基配对核苷酸上的胺取代更快。由于增加了局部灵活性，酰化专用于不匹配的2‘-胺基团。2‘-胺取代的探针寡核苷酸和靶序列都可以是DNA或RNA。该方法在查询错配和定量核酸群体中具有单碱基特异性的杂交方面具有广泛的潜在用途。此外，活化剂是一种双功能分子，其中突变敏感的酰化化学连接到一个独立的可视化部分。这个职业奖的目标是，首先，从根本上探索这种化学传感器技术的应用、机理和局限性，然后，开发一种实用的方法来可视化人类基因的突变。为新课程提供了详细的计划，并提出了创造性的想法来激励和挑战学生。

In this research, the PI will use a new chemical method that has been developed to detect point mutations and other defects in nucleic acid sequences. The method employs oligonucleotide probes in which one 2'-ribose position (-H or -OH in DNA or RNA, respectively) is substituted with an amine (-NH2) group. 2'-amine substituted nucleotides are specifically acylated by succinimidyl esters to form a 2'-amide product. The mutation detection method exploits an unanticipated observation by the PI that 2'-amine groups at the site of a mismatch are acylated more rapidly than amine substitutions at base paired nucleotides. Acylation is specific for mismatched 2'-amine groups due to increased local flexibility. Both the 2'-amine substituted probe oligonucleotide and the target sequence may be either DNA or RNA. The method has wide potential usefulness to query mismatches and quantify hybridization with single base specificity in nucleic acid populations. Moreover, the activated reagent is a bi-functional molecule in which the mutation-sensitive acylation chemistry is linked to an independent visualization moiety. Objectives of this Career Award are, first, to explore in a fundamental way the applications, mechanism, and limitations of this chemical sensor technology, and, thereafter, to develop a practical approach for visualizing mutations in human genes. The PI has proposed new experiments for an Advanced Biochemical Laboaratory including research based experiments and a bioinformatics module, and has established a literature based course on enzyme mechanisms.9984289WeeksIn this proposal, the PI will use a new chemical method that has been developed to detect point mutations and other defects in nucleic acid sequences. The method employs oligonucleotide probes in which one 2'-ribose position (-H or -OH in DNA or RNA, respectively) is substituted with an amine (-NH2) group. 2'-amine substituted nucleotides are specifically acylated by succinimidyl esters to form a 2'-amide product. The mutation detection method exploits an unanticipated observation by the PI that 2'-amine groups at the site of a mismatch are acylated more rapidly than amine substitutions at base paired nucleotides. Acylation is specific for mismatched 2'-amine groups due to increased local flexibility. Both the 2'-amine substituted probe oligonucleotide and the target sequence may be either DNA or RNA. The method has wide potential usefulness to query mismatches and quantify hybridization with single base specificity in nucleic acid populations. Moreover, the activated reagent is a bi-functional molecule in which the mutation-sensitive acylation chemistry is linked to an independent visualization moiety. Objectives of this Career Award are, first, to explore in a fundamental way the applications, mechanism, and limitations of this chemical sensor technology, and, thereafter, to develop a practical approach for visualizing mutations in human genes. Detailed plans are provided for new courses, and creative ideas are proposed to stimulate and challenge students.