基因工程的工具酶(一).pptVIP

第四讲　基因工程的工具酶 Enzymes for Gene Engineering DNase I足迹法（DNase I footprinting assay） 蛋白质结合在DNA片段上，能保护结合部位不被DNase破坏，DNA分子经酶切作用后遗留下该片段(亦称“足迹”)，进而可以确定它的序列。 平头双链DNA片段的连接操作 从分子动力学的角度讲，由限制性核酸内切酶创造的粘性末端的连接属于分子内部的连接，而平头末端的连接则属于分子间的连接，因此后者反应速度要慢的多。 提高平头末端连接效率的方法包括： 加大连接酶用量（10倍大于粘性末端的连接）。 加大平头末端底物的浓度，增加分子间碰撞机会。 加入10%PEG8000，促进大分子之间的有效作用。 加入单价阳离子（NaCl）,最终浓度150-200 mmol/L。 1)DNA连接酶不能催化两单链DNA分子连接； 2)只能连接双链DNA分子的单链缺刻(nick)； 3)不能连接双链中一个或多个核苷酸缺失所致的缺口（gap）。 DNA Polymerase I (or Pol I) is an enzyme that participates in the process of DNA replication in prokaryotes. It is composed of 928 amino acids, and is an example of a processive enzyme - it can sequentially catalyze multiple polymerisations. Discovered by Arthur Kornberg in 1956, it was the first known DNA polymerase (and, indeed, the first known polymerase of any kind). It was initially characterized in E. coli, although it is ubiquitous in prokaryotes. In E. coli and many other bacteria, the gene which encodes Pol I is known as polA. T4 DNA聚合酶(T4 phage DNA polymerase) 　 The activities of T4 DNA polymerase are very similar to Klenow fragment of DNA polymerase I - it functions as a 5 - 3 DNA polymerase and a 3 - 5 exonuclease, but does not have 5 - 3 exonuclease activity. The 3 - 5 exonuclease activity of T4 DNA polymerase is roughly 200 times that of Klenow fragment, making it preferred by many investigators for blunting DNAs with 3 overhangs. Taq polymerase is a thermostable DNA polymerase named after the thermophilic bacterium Thermus aquaticus from which it was originally isolated by Thomas D. Brock in 1965. One of Taqs drawbacks is its relatively low replication fidelity. It lacks a 3 to 5 exonuclease proofreading activity, and has an error rate measured at about 1 in 9,000 nucleotides. Some thermostable DNA polymerases have been isolated from other thermophilic bacteria and archaea, such as Pfu DNA polymerase, possessing a proofreading activity, and are being used instead of (or in combination with) Taq for high-fidelit