ENGINEERING AN IMPROVED THERMOSTABLE LIGASE

设计改进的热稳定连接酶

• 批准号: 6300472
• 负责人: FRANCIS BARANY
• 金额: $ 21.96万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-16 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6300472
• 关键词: DNA footprinting; DNA repair; biomarker; diagnosis design /evaluation; enzyme substrate; gene mutation; neoplasm /cancer diagnosis; nucleic acid sequence; phosphoester ligase; protein engineering; protein sequence; site directed mutagenesis; thermostability

(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.

（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。提高了特异性 通过详细分析突变体连接酶和修饰的底物。我们 正在开发一种生化和分子生物学方法 Thermus连接酶的保真度。 我们的具体目的是：（i）构建突变体热稳定连接酶和 测试更特异性。 TTH连接酶的位置定向诱变 将在通过肽序列数据识别的位点进行 从热的裂解和蛋白质序列比对获得 来自四个同源组的连接酶。 （ii）测试野生的特异性 类型和突变体可热稳定连接酶，用于区分底物 包含不同长度的单核苷酸重复。野生型和突变体 连接酶将被筛选，以检测单声道变化的能力 核苷酸重复。 （iii）测试改良的寡核苷酸以提高 连接期间的特异性。 各种接近3'端Q的效果 将评估有关连接保真度的类似物和行动条件 优化。 （iv）确定附加的序列和特异性 热稳定连接酶。 Thermus中的四个地理同源组 根据九个塔吉的序列比较提出了物种 Isoschizomers。 来自三个同源组的连接酶基因（美国，葡萄牙， 新西兰将进行测序，并将其与TTH DNA连接酶进行比较 代表日本集团。相应的新“ ISO-ligases”将是 过表达和表征。