生物化学与分子生物学学习课件：DNA重组和重组DNA技术 1.pptVIP

* Reverse transcriptase An enzyme first isolated from viruses: it will form DNA from an RNA templte, allowing complementary, or copy, DNA (cDNA) to be formed from mRNA. This allows double stranded cDNA sequences to be inserted into suitable plasmid vector, which can then be used to transform bacterial cells. Terminal transferase Catalyses the addition of nucleotides to the 3 terminus of a DNA molecule. It does not require a template. The preferred substrate of this enzyme is a 3-overhang, but it can also add nucleotides to blunt or recessed 3 ends. DNA polymeraseⅠ DNA Polymerase I : prokaryotic DNA polymerase in DNA replication. Pol I possesses three enzymatic activities: A 5’-3’ (forward) DNA polymerase activity. A 3 - 5 (reverse) exonuclease activity that mediates profreading. A 5 - 3 (forward) exonuclease activity mediating nick translation during DNA repair. Klenow fragment The Klenow fragment is a large protein fragment produced when DNA polymerase I from E. coli is enzymatically cleaved by the protease subtilisin. It retains the 5-3 polymerase activity and the 3’ → 5’ exonuclease activity for removal of precoding nucleotides and proofreading, but loses its 5 → 3 exonuclease activity. Taq DNA polymerase Heat stale DNA polymerase form Thermus aquaticus , a bacterium that lives in hot springs It is able to withstand the protein-denaturing conditions (high temperature) required during PCR 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 T4 polynucleotide kinase Catalyzed the transfer of Terminal (γ) phosphate from a nucleotide 5’ triphosphate to a 5’ hydroxyl group of a polynucleotide. 二、重组DNA技术中常用的载体 定义 为携带目的基因，实现其无性繁殖或表达有意义的蛋白质所采用的一些DNA分子。 载体按功能分为 克隆载体(cloning vector) 表达载体(expression vector) 克隆载体(cloning vector) 为使插入的外源DNA序列被扩增而特意设计的载体称为克隆载体。 表达载体(expression vector) 为使插入的外源DNA序列可转