ENGINEERING AN IMPROVED THERMOSTABLE LIGASE

(申请人的描述)热稳定Tth DNA的特异性 连接酶在确定 PCR/LDR 的灵敏度和 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 栖热菌连接酶的保真度。 我们的具体目标是：（i）构建突变型热稳定连接酶和 测试以获得更大的特异性。 Tth 连接酶的定点诱变 将在肽序列数据识别的位点上进行 通过 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- 用于区分底物的类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链变化的能力 核苷酸重复。 (iii) 测试修饰的寡核苷酸是否具有更大的 连接过程中的特异性。 各种近3'端Q的影响 将评估连接保真度的类似物并确定作用条件 优化。 (iv) 确定附加的序列和特异性 热稳定性连接酶。栖热菌中的四个地理同源群 根据九个 TagI 的序列比较提出了物种 同裂酶。 来自三个同源组（美国、葡萄牙、 新西兰）将被测序并与 Tth DNA 连接酶进行比较， 代表日本组。相应的新“异连接酶”将是 过度表达和表征。

(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.