1、152上海海洋大学 食品学院 08 制药一班 OH 翻译版I6 POLYMERASE CHAIN REACTION (PCR)【聚合酶联反应】Key NotesPrinciples of PCR【聚合酶联反应的原理 】 The polymerase chain reaction (PCR) allows an extremely large number of copies to be synthesized of any given DNA sequence provided that two oligoonucleotide primers are available that hybri
2、dize to the flanking sequences on the complementary DNA strands. 【聚合酶联反应指一种在体外快速扩增特定基因或 DNA 序列的方法】The reaction requires the target DNA, the two primers, all four deoxyribonucleoside triphosphates and a thermostable DNA polymerase such as Taq DNA polymerase. 【这种反应需要目标 DNA，两个引物，四个脱氧和干三磷酸和一个耐热 DNA 聚合酶例
3、如噬热水生菌 DNA 聚合酶】A PCR cycle consists of three steps; denaturation, primer annealing and elongation.【一个 PCR 循环反应包括 3 个步骤：模板变性】 This cycle is repeated for a set number of times depending on the degree of amplification required.【这个循环将重复一定的次数，而重复的次序是取决于需要扩增的程度】Applications of PCR【PCR 的程度】 PCR has made a
4、huge impact in molecular biology, with many applications in areas such as cloning, sequencing, the creation of specific mutations, medical diagnosis and forensic medicine.【PCR 技术很大程度上影响了分子生物学的发展，在这个领域上有很大的应用例如克隆，测序，创造特定的基因突变，医疗诊断和法医学】Related topics DNA structure (F1) 【DNA 结构】 Nucleic acid hybridizat
5、ion (I2) 【核酸杂交】DNA replication in prokaryotes (F3) 【在原核生物中的 DNA 复制】DNA cloning (I3) 【DNA 克隆】Restriction enzymes (I1) 【限制性内切酶】 DNA sequencing (I5) 【DNA 测序】Principles of PCR【PCR 的原理】PCR (polymerase chain reaction) is an extremely simple yet immensely powerful technique. 【PCR( 聚合酶联反应)一种操作简单，应用强大的技术 】It
6、 allows enormous amplification of any specific sequence of DNA provided that short sequences either side of it are known. The technique is shown in Fig.15. 【PCR满足任何特定序列的 DNA 哪怕这条 DNA 只有一端是一直的】A PCR reaction contains the target double-stranded DNA, two primers that hybridize to flanking sequences on
7、opposing strands of the target, all four deoxyribonucleoside triphosphates and a DNA polymerase. 【PCR 反应包括目标双链 DNA，用于杂交目标链的反向链开始杂交的两个引物，四种脱氧核苷酸三磷酸和一个 DNA 聚合酶】Because, as we shall see, the reaction periodically gets heated to high temperature, PCR depends upon using a heat-stable DNA polymerase. 【因此，
8、正如我们将会看到的那样，加热到如期的温度时反应开始进行， PCR 开始通过一个热稳定的 DNA 聚合酶开始起作用】Many such heat-stable enzymes from thermophilic bacteria (bacteria that live in high temperature surroundings) are now available commercially. 【拿下许多来自嗜热细菌（那些喜欢生活在高温环境下的细菌）的热稳定酶现在运用的很广泛】The first one used was Taq polymerase from the thermophili
9、c bacterium Thermus aquaticus. 【第一个用来作 DNA 聚合酶的是来自于水生嗜热菌的 Taq 聚合酶】PCR consists of three steps:【PCR 包括以下三种步骤】denaturation. 【变性】The reaction mixture is heated to 95 for a short time period (about 15-30 sec) 153to denature the target DNA into single strands that can act as templates for DNA synthesis.【
10、将反应混合物加热到 95 摄氏度并保持短暂的时间（大约是 1530 秒钟）目标 DNA 就会变性必恩威单链DNA 然后作为 DNA 合成的模板】Primer annealing. 【引物退火 】The mixture is rapidly cooled to a defined temperature which allows the two primers to bind to the sequences on each of the two strands flanking the target DNA. 【将混合物迅速冷却到特定的温度，这个特定的温度可以将两个引物从目标 DNA 的两端开
11、始连接】This annealing temperature is calculated carefully to ensure that the primers bind only to the desired DNA sequences. 【这个退火温度是经过仔细计算而得的，它可以确保引物只和设定好的 DNA 联合】One primer binds to each strand (Fig .1). 【正如图一所示一个引物从一端开始连接 】The two parental strands do not reanneal with each other because the primers
12、are in large excess over parental DNA.【两条母链不会相互联合起来因为母链的 DNA 引物是过量的】Elongation. 【延伸】 The temperature of the mixture is raised to 72 (usually) and kept at this temperature for a pre-set period of time to allow DNA polymerase to elongate each primer by copying the single-stranded templates.【混合物的温度在升到
13、72 摄氏度并保持预先设定的温度把持一段时间，这是通过复制单链模板让 DNA 聚合酶延伸每个引物】 Thus at the end of this incubation, both single-stranded template strands have been made partially double stranded. 【然后，培养结束了之后，每个单链模板会合成双链】The new strand of each double-stranded DNA extends for a variable distance downstream.【每条双链 DNA 的新联会延长并成为下一步的变
14、量】The three steps of the PCR cycle are repeated. 【PCR 的这三个步骤是循环重复的 】Thus in the second cycle, the four strands denature, bind primers and are extended.【因此，在第二步骤中，就会有四条例案变性，连接引物然后再延伸】 No other reactants need to be added.【没有别的步骤需要被增加】 The three steps are repeated once more for a third cycle (Fig.15.)
15、and so on for a set number of additional cycles. 【这三个步骤一旦重复三次之后在设置额外的循环数目】Automated thermocyclers are now routinely used to cycle the reaction without manual interference. 【现在通常使用“热循环”来完成反应，而不是考人工来控制】After 20 cycles, the original DNA has been amplified a million-fold and this rises to a billion-fold
16、 (1000 million) after 30 cycles.【在 20 个循环之后，源 DNA 就可以大量的复制，在 30 个循环之后 DNA 的数目会增加到 1000 万】 All of this takes less than an hour. 【这些可以在一消失内完成】The vast majority of the products are identical in that the DNA amplified is that between the two primer binding sites and does not extend beyond these sites. 【
17、绝大多数的产品是相同的 DNA 在两个引物之间复制不会超过这些范围】Only in the first few cycles do longer DNA molecules represent a significant fraction of the total product.【只有在一开始的几个循环中较长的 DNA 分子代表全部的产品】Applications of PCR【PCR 的应用】PCR already has very widespread applications, and new uses are being devised on a regular basis. Som
18、e (and certainly not all) of the applications are as follows: 【PCR 技术现在已经被大量的应用，新的应用还在深入到更加基层的技术，下面举了几个应用的例子】PCR can amplify a single DNA molecule from a complex mixture, largely avoiding the need to use DNA cloning to prepare that molecule.【PCR 可以大量的在一个混合物中大量复制单链 DNA分子，大幅度的避免了使用 DNA 克隆的方法准备分子】 Vari
19、ants of the technique can similarly amplify a specific single RNA molecule from a complex mixture.【很多技术可以像 PCR相似，复制大量的在复杂的混合物种复制特定的单链 DNA 分子】DNA sequencing has been greatly simplified using PCR, and this application is now 154common.【DNA 序列可以用 PCR 技术大量的被复制冰雪这种复制的方法现在很普遍】By using suitable primers, it
20、 is possible to use PCR to create point mutations, deletions and insertions of target DNA which greatly facilitates the analysis of gene expression and function.【只要有合适的引物，就可以应用 PCR 技术创造点突变，变性并且转染需要被基因表达和功能分析的目标 DNA】PCR is exquisitely sensitive and can amplify vanishingly small amounts of DNA. 【PCR 很
21、灵敏，它可以在不知不觉中放大少量的 DNA】Thus, using appropriate primers, very small amounts of specified bacteria and viruses can be detected in tissues, making PCR invaluable for medical diagnosis.【因此，应用合适的引物，少量的特定的细菌和病毒就可以在组织中被检测到，这是用 PCR 技术用于疾病的诊断 】PCR is now invaluable for characterizing medically important DNA s
22、amples. 【PCR 技术目前是医学上重要的确定 DNA 样本的技术】For example, in screening for human genetic diseases, it is rapidly replacing the use of RFLPs (see Topic I1). 【举个例子，在筛选人类基因疾病的时候可以应用 PCR 技术来代替 RFLP 技术（详细见 I1 主题） 】The PCR screen is based on the analysis of microsatellites. 【PCR 筛选是用于在微卫星的分析中】These are di-, tri-
23、and tetranucleotide repeats in the DNA of the type (CA)n or (CCA) n where n is a number from 10 to more than 30. 【那些双核苷酸，三核苷酸，四核苷酸可以在（CA）n 或者是（CCA）n（n 的范围是 1 到30 以上）的 DNA 里复制】The microsatellites can be used as markers in the same way that RFLPs were used in the past (see Topic I1). 【在过去，微卫星通常使用 PFLP
24、s 做标记】Thus two primers are chosen that bind to the DNA flanking the microsatellite. 【然后选两个引物在微卫星中用来连接 DNA 两端】PCR is then carried out and the different sizes of microsatellite give different sizes of amplified DNA fragments that can then be used as screening markers.【PCR 允许不同型号的微卫星给予的不同的大小的 DNA 片段并用来
25、作为筛选的记号】 The method is very fast, reliable and uses very small amounts of clinical material.【这个方法很快，可信度很高并可以用于临床数据很少的案例中】Because of its extreme sensitivity, PCR is now fundamentally important to forensic medicine. 【由于 PCR 有高度的敏感性，所以这项技术在法医鉴定这一方面有很大的帮助】It is even possible to use PCR to amplify the DNA from a single human hair or a microscopic drop of blood left at the scene of a crime to allow detailed characterization. 【法医可以利用 PCR 技术，从犯罪现场所搜集来的头发或血液中扩增出 DNA 来进行罪犯的确定】
