PCR扩增DNA

【实验目的】
1.掌握PCR扩增DNA的技术及原理
2.学习PCR扩增仪的使用

【实验原理】
聚合酶链反应（polymerase chain reaction，PCR）是一种体外DNA扩增技术，1985年由Mullis等人创立。该技术能在几小时的实验操作中，将人为选定的一段DNA扩增几百万倍，具有灵敏度高、特异性强、操作简便和应用广泛等优点，目前已成为分子生物学及基因工程中极为有用的研究手段，另外在医学研究和医疗诊断中亦体现出极大的应用价值。
PCR的工作原理类似于DNA的体内复制过程，是将待扩增的DNA片断和与其两侧互补的寡核苷酸链引物经变性、退火及延伸三步反应的多次循环，使特定的DNA片断在数量上呈指数增加。PCR扩增首先需要一对引物，根据待扩增区域两侧的已知序列合成两个与模板DNA互补的寡核苷酸作为引物，引物序列将决定扩增片段的特异性和片段长度。反应体系由模板DNA、一对引物、dNTP、耐高温的DNA聚合酶、酶反应缓冲体系及必须的离子等所组成。PCR反应循环的第一步为加热变性，使双链模板DNA变性为单链；第二步为复性，每个引物将与互补的DNA序列杂交；第三步为延伸，在耐高温的DNA聚合酶作用下，以变性的单链DNA为模板，从引物3ˊ端开始按5ˊ→3ˊ方向合成DNA链。这样经过一个周期的变性——复性——延伸等三步反应就可以产生倍增的DNA，假设PCR的效率为100%,反复n周期后，理论上就能扩增2n倍。PCR反应一般30-40次循环，DNA片段可放大数百万倍。
常规PCR反应用于已知DNA序列的扩增，变性温度为95℃，复性温度为37℃～55℃，延伸合成温度为72℃，DNA聚合酶为Taq酶（可耐受95℃左右的高温而不失活），反应循环数为30。

【实验材料】
1.实验器材
PCR扩增仪，台式高速离心机，移液器，经高压灭菌后的Eppendorf管，电泳仪。
2.实验试剂
（1）模板DNA（0.1µg/µl）：用牙签挑取单菌落悬浮到50µl的裂解液中（裂解液1%TritonX-100，20mmol/l Tris-HCl PH 8.2, 2mmol/l EDTA）, 95℃温浴10分钟, 10000rpm离心5分钟,取2µl上清液用于总体积50µl的PCR反应。
（2）TaqDNA聚合酶（5U/µl），10×扩增缓冲液，dNTP（1mmol/L）：购买
（3）引物（100pmol/L）：设计并合成与目的DNA两侧互补的引物内。

【实验操作】
1．准备PCR反应溶液
（1） 按以下次序，将下列成分在0.5ml灭菌Eppendorf管内混合：
10×扩增缓冲液 10µl
4×dNTPs(包括四种dNTP) 8µl
引物1 1µl
引物2 1µl
模板DNA 1µl (10ng)
Taq DNA聚合酶 1µl (2.5U)
加水至终体积 100µl
（2） 用手指轻弹Eppendorf管底部，使溶液混匀。在台式离心机中离心2秒以集中溶液于管底。
（3）加石蜡油50µl封住溶液表面。

PCR Amplify DNA

【Purpose】
1.Master the principle and the techniques of PCR Amplifying DNA
2.Learn how to use the PCR thermal cycler

【Principle】
Ploymerase chain reaction (PCR) is a technique of amplifying DNA in vitro, which was previously established in 1985 by Mullis et al. The technique, by which a fragment of DNA can be amplified many million-fold in a few of hours under experimental operation, has the advantages of high sensitivity，high specificity, ease of operation, extensive application and so on. Until now PCR has become an essential tool not only in Molecular Biology but also in genetic engineering. Furthermore，its applications are finding their way into many areas of medical research and medical diagnosis.
The principle of PCR is similar to the natural process of DNA replication. The DNA fragment is amplified with two oligonucleotide primers，each complementary to one end of the DNA target sequence，in a three-step reaction of denaturing, annealing and polymerization. Many cycles can produce the definite DNA fragments increased in quantity by index. A pair of oligonucleotides will serve as PCR primers, which can be designed according to the known sequence information of template DNA .The primers are complementary to one end of the DNA target sequence so that they can decide the length and the location of the amplified DNA fragment. The reaction system comprises template DNA, a pair of primers, dNTPs, thermostable DNA polymerase, reaction buffer，magnesium and optional additives. Each cycle of PCR is as follows: Firstly , denaturation— Heat to denature the duplex DNA template at high temperature so that the target DNA is separated into two strands; Secondly, annealing— Decrease the temperature of the solution to make the primers and DNA template complement and anneal to partly form double chains; Finally, extension—With thermostable DNA polymerase , the single strand of the DNA template, which is obtained by denaturation, distends to be double strands by addition of nucleotides to primer ,s 3ˊend. As a result, by reaction cycle of three steps: denaturation, primer annealing and polymerization ,the copy of the definite DNA fragment is doubled over and over again with each cycle. If PCR is 100% efficient, one target molecule would become 2n after n cycles. Usually by 30～40 cycles, DNA fragment is amplified by some million times.