PCR 多聚酶链式反应 生命科学前沿 教学课件.pptVIP

三 PCR技术 PCR (Polymerase chain reaction) 是用一对寡聚DNA作为引物，通过加温变性－退火－DNA合成这一周期的多次循环，使目的DNA片段得到扩增。由于这种扩增产物是以指数形式积累的，经25－30个循环后，扩增倍数可达106。 Dr. Karry Mullis 1985年发明，获1993年度诺贝尔化学奖； pfu DNA 聚合酶 耐热 5’?3’DNA聚合酶活性 3’→5’外切酶活性 精确度：pfu Taq 但pfu扩增效率通常比Taq酶略差 文献报道：Taq+pfu 会起到较好效果 手工设计引物的人似乎不多，还是用软件方便些，防止你一不小心看走眼，丢一个碱基，同时计算起来也方便。设计软件有很多，既可以在线设计（如Primer3 /cgi-bin/primer3/primer3_www.cgi），也可以用Primer5、Oligo6.65 等等。细心地进行引物设计是PCR 中最重要的一步。理想的引物对只同目的序列两侧的单一序列而非其他序列退火。 设计糟糕的引物可能会同时扩增其他的非目的序列。 100ng 到1μg 的人类基因组DNA，相当于3×104 到3×105 个分子。 PCR特异性依赖于与靶序列 两端互补的寡核苷酸引物。 半保留复制链重复循环变性--退火--延伸 三过程，就可获得更多的“半保留复制链”，而且这种新 链又可成为下次循环的模板。每完成一个循环需2～4分 钟， 2～3小时就能将待扩目的基因扩增放大几百万倍。 到达平台期(Plateau)所需循环次数取决于样品中模板的拷贝。 DNA amplification by the polymerase chain reaction (PCR) is schematically outlined in the diagram to the left. There are 3 general steps to the process that are repeated for a number of cycles to exponentially increase the number of copies of a specific target region. Genomic DNA is normally double-stranded (DS-DNA).?? STEP 1 is to first unzip the DS-DNA, also called denaturation, into two complementary single strands of DNA by heating the reaction mix to 95 degrees Celsius. STEP 2 isolates the target region of the Genomic DNA by landing 2 primers (P1 P2) which exactly match two 20-30 unique base pair regions that bookend the target region. This is called annealing. Once time is allowed for the primers to land on the sites, STEP 3 invloves heating the mix to 72 degrees at which point a special polymerase builds the DNA strand starting at the primers and continuing in the 5 prime direction. This is called extension.?? These three steps are repeated 25-40 times to produce millions of exact copies of the target region of DNA. Because during the second cycle of this process, extension can occur on both the original copy of genomic DNA and the newest pieces (the colored ones in the diagram) subsequent extensions are quickly limited precisely to the target region (A). 反应最终的DNA 扩增量可用Y＝(1＋X)n计算。Y代表D