Evolution of a Deoxyribozyme Ligase

描述(申请人提供)：DNA作为稳定催化剂的潜力可能使该分子在分子生物学、生物技术、纳米技术和临床诊断方面比核酶更成功。此外，比较自然和更频繁地出现的催化RNA结构和产生催化基序的DNA结构将被证明是揭示生物催化剂的基本特征的关键。为了进一步了解核酸酶的进化及其结构和功能，这项建议的目标是1)使用逐步进化过程从随机序列进化出脱氧核酶连接酶，2)优化脱氧核酶催化的连接酶链式反应(LCR)，这是一种通过连接底物和模板的变性和退火循环来指数扩增目标DNA的方法，以及3)使用脱氧核酶催化的LCR检测遗传突变，如BRCA1、BRCA1的点突变和导致β-地中海贫血的突变。

DESCRIPTION (provided by applicant): The potential of DNA as a stable catalyst may give this molecule more success than ribozymes as tools for molecular biology, biotechnology, nanotechnology, and clinical diagnostics. Furthermore, comparison of catalytic RNA structures, which occurs naturally and more frequently, and DNA structures that result in catalytic motifs will prove crucial in revealing fundamental features of biocatalysts. To further gain insight into evolution and the structure and function of nucleic acid enzymes, this proposal will aim to 1) evolve a deoxyribozyme ligase from random sequences using stepwise evolution procedures, 2) optimize a deoxyribozyme-catalyzed ligase chain reaction (LCR), a method for the exponential amplification of target DNA through cycles of denaturing and annealing of ligation substrates and template, and 3) detect genetic mutations using the deoxyribozyme-catalyzed LCR, such as point mutations of BRCA1, BRCA1, and ones that give rise to beta-thalassemia.