南开大学基因操作原理第三章.ppt

* * * * * * * * * * * * * * * * * * * 绝大多数的Ⅱ型限制性核酸内切酶都能够识别由4-8个核苷酸组成的特定的核苷酸序列。 限制性核酸内切酶就是从其识别序列内切割DNA分子的，因此这些识别序列又叫核酸内切酶的切割位点或靶序列。 在 DNA 分子双链的特异性识别序列部位，切割 DNA 分子，产生链的断裂。 2 个单链断裂部位在 DNA 分子上的分布，通常不是彼此直接相对。断裂结果形成的 DNA 片段，具有互补的单链延伸末端。 * * * * * * * * * * * * * * * * * * * * Reaction system：enzyme, substrate, buffer… 1U：in most appropriate reaction conditions, 60 min, 1μg DNA * Activity factor: temperature, buffer, time, reaction volume, Glycerol concentration, DNA purity and structure eg： 4 μg DNA / 1U HpaⅠ 50 μl 37℃ 15 h 1 μg DNA / 1U HpaⅠ 50 μl 37℃ 1 h Reaction conditions of restriction enzymes II Mechanism of DNA Ligase Characteristic and application of DNA Ligase * 3. DNA Ligase DNA ligase: E.coli and T4 phage can encode an enzyme, which seals single-stranded nicks between adjacent nucleotides in a duplex DNA chain. * * 1) Mechanism of DNA ligase Characteristic of DNA ligase (1) 5’ … G-C-T-C-T-G-C-A G-G-A-G … 3’ 3’ … C-G-A-G A-C-G-T-C-C-T-C … 5’ OH P P OH 5’ … G-C-T-C-T-G-C-A-G-G-A-G … 3’ 3’ … C-G-A-G-A-C-G-T-C-C-T-C … 5’ DNA Ligase nick nick * (2) nick 5’ … G-C-U-C-U-G-C-C-G-G-A-G … 3’ 3’ … C-G-A G-A-C-G-G-C-C-T-C … 5’ OH P 5’ … G-C-U-C-U-G-C-C-G-G-A-G … 3’ 3’ … C-G-A-G-A-C-G-G-C-C-T- C … 5’ DNA Ligase * (3) 5’ … G-C-T-C-A-G-OH P-C-T-G-G-A-G … 3’ 3’ … C-G-A-G-T-C-P OH-G-A-C-C-T-C … 5’ 5’ … G-C-T-C-A-G-C-T-G-G-A-G … 3’ 3’ … C-G-A-G-T-C-G-A-C-C-T-C … 5’ DNA Ligase * T4DNA Ligase T4 DNA Ligase－－ATP Cohesive end Blunt end E.coli DNA Ligase DNA Ligase －－NAD+ Cohesive end 　　 * Type of DNA Ligase * * ——the optimum temperature: 370C but at this temperature the hydrogen-bonded join between the sticky ends is unstable, so the temperature is a compromise between the enzyme action and association of the termini, the optimum temperature is 160C. * 2) Characteristic and application of DNA ligase Amount of T4 DNA Ligase： Blunt end: 1～2 U Cohesive end: 0.1 U ATP: 10 μmol~1 mmol/L