ENGINEERING AN IMPROVED THERMOSTABLE LIGASE

（申请人的描述）热稳定Tth DNA的特异性 连接酶在决定PCR/LDR的灵敏度方面起重要作用， PCR/RE/LDR检测系统。获得了更高的特异性 通过对突变连接酶和修饰底物的详细分析。我们 正在开发一种生物化学和分子生物学方法来研究 Thermus连接酶的保真度。 我们的具体目标是：（i）构建突变型热稳定连接酶， 测试更大的特异性。Tth连接酶的定点突变 将在肽序列数据确定的位点上进行 从Thermus的亲和切割和蛋白质序列比对获得 来自四个同源组的连接酶。 (ii)测试野生动物的特异性- 用于区分底物的型和突变型热稳定连接酶 含有不同长度的单核苷酸重复序列。野生型和突变型 将筛选连接酶检测单- 核苷酸重复序列。 (iii)测试修饰的寡核苷酸， 在连接期间的特异性。 不同的近3 '-端Q值 将评估类似物对连接保真度的影响， 优化 (iv)确定额外的序列和特异性 热稳定连接酶栖热菌属的四个地理同源群 根据9个TagI的序列比较，提出了10个新的物种 异构体。 来自三个同源组的连接酶基因（美国，葡萄牙， 新西兰）进行测序，并与Tth DNA连接酶进行比较， 代表日本集团。相应的新“异连接酶”将是 过度表达和特征化。

(Applicant's description) The specificity of the thermostable Tth DNA ligase plays a vital role in determining the sensitivity of PCR/LDR and PCR/RE/LDR detection systems. Increased specificity has been acquired through detailed analysis of mutant ligases and modified substrates. We are developing a biochemical and molecular biological approach to study the fidelity of Thermus ligases. Our specific aims are: (i) Constructing mutant thermostable ligases and testing for greater specificity. Site-directed mutagenesis of Tth ligase will be conducted on sites identified by the peptide sequence data obtained from affinity cleavage and protein sequence alignment of Thermus ligases from four homology groups. (ii) Testing the specificity of wild- type and mutant thermostable ligases for discriminating substrates containing different length mono-nucleotide repeats. Wild-type and mutant ligases will be screened for their ability to detect changes in mono- nucleotide repeats. (iii) Testing modified oligonucleotides for greater specificity during ligation. The effect of various near-3'-end Q analogues on ligation fidelity will be evaluated and action conditions optimized. (iv) Determining the sequence and specificity of additional thermostable ligases. Four geographic homology groups among Thermus species were proposed based on sequence comparisons of nine TagI isoschizomers. Ligase genes from three homology groups (USA, Portugal, New Zealand) will be sequenced and compared with the Tth DNA ligase which represents the Japan group. The corresponding new "iso-ligases" will be over-expressed and characterized.