ENGINEERING AN IMPROVED THERMOSTABLE LIGASE

(申请人描述)耐热TTH DNA的特异性 连接酶在决定PCR/LDR的敏感性中起着至关重要的作用 PCR/RE/LDR检测系统。已经获得了更高的专一性 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的方法来研究 Thermus连接酶的保真度。 我们的具体目标是：(I)构建突变的耐热连接酶和 测试更大的特异性。TTH连接酶的定点突变 将在由肽序列数据识别的位置上进行 从Thermus的亲和力切割和蛋白质序列比对中获得 来自四个同源基团的连接酶。(Ii)测试野生- 用于区分底物的耐热连接酶类型和突变 包含不同长度的单核苷酸重复。野生型和突变型 将对连接酶进行筛选，以了解其检测单核苷酸变化的能力。 核苷酸重复。(3)检测修饰的寡核苷酸是否具有更高的 结扎过程中的特异性。不同近3‘端Q的影响 将评估关于连接保真度的类似物和作用条件 最优化。(四)确定附加条款的先后顺序和特殊性 耐热连接酶。Thermus间的四个地理同源群 根据9个TAGI的序列比较，提出了物种 等裂解构体。三个同源组的连接酶基因(美国、葡萄牙、 新西兰)将被测序，并与TTH DNA连接酶进行比较 代表日本队。相应的新“异连接酶”将是 过度表达和刻画。

(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.