PCR实验报告(分子生物学实验).pdf

PCR Lin Chengyu Bio 04 2010030007; Cooperator: Yuan Xiaowen Experiment date: 2012-03-22 Submit Date: 2012-03-23 1 Introduction 1.1 Background information PCR (polymerase chain reaction) is a technique for amplifying DNA sequences in vitro. It was invented by Kary Mullis and his colleagues in 1985. It is widely used in gene cloning, mutation, sequencing and detection. 1.2 Objectives (1) Learn the principle and method of PCR (Polymerase Chain Reaction). (2) Comprehend the significance of PCR technique in DNA manipulation. 1.3 Major principles One typical PCR cycle consists of three steps: denaturation, annealing and extension. (1) Denaturation Figure 1 Step 1: denaturation As shown in Figure 1, the target double-strand DNA can be separated into single-strand DNA under high temperature (about 50 ℃). (2) Annealing Figure 2 Step 2: annealing As shown in Figure 2, when temperature is lower, the forward and reverse primers will bind to the single strand DNA, which are about 20 nt long, designed to be complementary to the both end of the target gene and often has restriction enzyme recognition sites at the 5 ’ends. (3) Extension Figure 3 Step 3: extension As shown in Figure 3, when temperature rise to about 72 ℃, which is the optimal temperature of Taq, one kind of DNA polymerase working in high temperature. The discovery of Taq DNA polymerase is fundamental to PCR. As a result, new DNA strand complementary to the