PCR聚合酶鏈反應(yīng)英語(yǔ)介紹

1、VirologyThe BasicsTaxonomyLinksHaematologyDNA Down UnderPCR Down UnderHOME HeadlinesDNA Down UnderNew section on RNA interference as a tool to block virus replication.The polymerase chain reaction (PCR) is a technique for copying a piece of DNA a billion-fold. As the name suggests, the process creat

2、es a chain of many pieces, in this case the pieces are nucleotides, and the chain is a strand of DNA. PCR is an enzyme-mediated reaction, and as with any enzyme, the reaction must occur at the enzyme's ideal operating temperature. The enzymes that are used for the PCR are DNA-dependent DNA polym

3、erases (DDDP) derived from thermophilic (heat-loving) bacteria. As such, the enzymes function at higher tempertaures than the enzymes we commonly use in the laboratory or have working in our bodies. These DNA polymerases operate at 60-75°C, and can even survive at temperatures above 90°C.

4、This is important because a part of the PCR requires that the reaction reaches 95°C as we shall see.Apart from the DNA polymerase, PCR needs a DNA template to copy, and a pair of short DNA sequences called oligonucleotides or "primers" to get the DNA polymerase started.Broadly speakin

5、g, there are three steps identifed by incubating at different tempratures. the three steps make up a PCR "cycle". 1 Double-stranded DNA separation or denaturation (D in Figure 1) 2 Primer annealing to template DNA (A in Figure 1) 3 Primer extension (E in Figure 1) Figure 1.A PCR cycle.The

6、three temperatures which make up a single cycle. The DNA denaturatiuon section (D), oligonucleotide annealing section (A) and the primer extension (E) section are marked. The temperature range over which dsDNA duplexes can denature (TD) or 'melt', and the range over which the oligonucleotide

7、 primer can hybridise (TM) are also marked.DenaturationAt temperatures above 90°C, double-stranded DNA denatures or "melts". That means the weak hydrogen bonds that usually hold the two complementary strands together at normal temperatures are disrupted resulting in two single strande

8、d DNA strands (shown below in an idealised form).Primer AnnealingAt the annealing temperature (TA), primers that collide with their complementary sequence can hybrdise or "bind" to it. The chance of such an encounter happening is increased because we use a vast excess of each primer in the

9、 reaction mixture compared to the number of template molecules present.The assay in the example below has been designed to amplify a region of the template spanned by and including, the primer sequences.Primer ExtensionAt the extension temperature (TE), the DNA polymerase binds to the hybridised pri

10、mer and begins to add complementary nucleotides (i.e. every time the polymerase reads a "G" on the template strand, its adds a "C" an "A" for a "T" a "G" for a "C" and a "T" for an"A"), chemically binding each new additi

11、on to the last to form a growing chain. The process only occurs in one direction. In our example, the green primer is binding to its complementary template sequence and is facing toward the right (this is called the 5' (five-prime) to 3' (three prime) direction. Extension occurs in the direc

12、tion that the primer faces. The result is a new double-stranded PCR product we usually call an "amplicon". An amplicon can be defined as an amplified molecule of a single type, in this case, an exact replicate of the original template.Exponential Template DuplicationThe process is then rep

13、eated by cycling through the tempratures over and over again (35 to 55 times). Each cycle results in a new DNA duplex, each strand acting as a potential template for one or other primer.Some interesting things stand out from the figure below. The original template strands (blue and red) continue to

14、act as templates because the PCR process is not destructive. However, each cycle produces a greater number of the shorter amplicon molecules. These are shorter in our example because the primers shown, bind within the template sequence. Eventually the majority of the amplicon in the recation vessel

15、will be the expected length, i.e. just the region spanned by, and including the primer sequences.It is possible to mathematically predict the pattern of amplicon accumulation. In our example, we have started with two strands. In a perfect PCR recation (which rarely occurs!), we have two new strands

16、making a total of four. After the second cycle we have eight strands, then 16, 32 and so on. The reaction is doubloing the nu,ber of strands each cycle opr to make that an equation, we have 2nNote: to make the process easier to understand, I have drawn the DNA strands as straight lines - in reality,

17、 DNA does not exist in as simple a form as this. | About VDU | Contact VDU |What is PCR (polymerase chain reaction)?PCR (polymerase chain reaction) is a method to analyze a short sequence of DNA (or RNA) even in samples containing only minute quantities of DNA or RNA. PCR is used to reproduce (ampli

18、fy) selected sections of DNA or RNA. Previously, amplification of DNA involved cloning the segments of interest into vectors for expression in bacteria, and took weeks. But now, with PCR done in test tubes, it takes only a few hours. PCR is highly efficient so that untold numbers of copies can be ma

19、de of the DNA. Moreover, PCR uses the same molecules that nature uses for copying DNA:· Two "primers", short single-stranded DNA sequences that are synthesized to correspond to the beginning and ending of the DNA stretch to be copied;· An enzyme called polymerase that moves along

20、 the segment of DNA, reading its code and assembling a copy; and· A pile of DNA building blocks that the polymerase needs to make that copy.How is PCR (polymerase chain reaction) done?As illustrated in the animated picture of PCR, three major steps are involved in a PCR. These three steps are r

21、epeated for 30 or 40 cycles. The cycles are done on an automated cycler, a device which rapidly heats and cools the test tubes containing the reaction mixture. Each step - denatauration (alteration of structure), annealing (joining), and extension - takes place at a different temperature:1 Denaturat

22、ion:At 94 C (201.2 F), the double-stranded DNA melts and opens into two pieces of single-stranded DNA.2 Annealing:At medium temperatures, around 54 C (129.2 F), the primers pair up (anneal) with the single-stranded "template" (The template is the sequence of DNA to be copied.) On the small

23、 length of double-stranded DNA (the joined primer and template), the polymerase attaches and starts copying the template.3 Extension:At 72 C (161.6 F), the polymerase works best, and DNA building blocks complementary to the template are coupled to the primer, making a double stranded DNA molecule.Wi

24、th one cycle, a single segment of double-stranded DNA template is amplified into two separate pieces of double-stranded DNA. These two pieces are then available for amplification in the next cycle. As the cycles are repeated, more and more copies are generated and the number of copies of the templat

25、e is increased exponentially.What is the purpose of doing a PCR (polymerase chain reaction)?To do PCR, the original DNA that one wishes to copy need not be pure or abundant. It can be pure but it also can be a minute part of a mixture of materials. So, PCR has found widespread and innumerable uses -

26、 to diagnose genetic diseases, do DNA fingerprinting, find bacteria and viruses, study human evolution, clone the DNA of an Egyptian mummy, establish paternity or biological relationships, etc. Accordingly, PCR has become an essential tool for biologists, DNA forensics labs, and many other laborator

27、ies that study genetic material.How was PCR (polymerase chain reaction) discovered?PCR was invented by Kary Mullis. At the time he thought up PCR in 1983, Mullis was working in Emeryville, California for Cetus, one of the first biotechnology companies. There, he was charged with making short chains

28、of DNA for other scientists. Mullis has written that he conceived of PCR while cruising along the Pacific Coast Highway 128 one night on his motorcycle. He was playing in his mind with a new way of analyzing changes (mutations) in DNA when he realized that he had instead invented a method of amplify

29、ing any DNA region. Mullis has said that before his motorcycle trip was over, he was already savoring the prospects of a Nobel Prize. He shared the Nobel Prize in chemistry with Michael Smith in 1993.As Mullis has written in the Scientific American: "Beginning with a single molecule of the gene

30、tic material DNA, the PCR can generate 100 billion similar molecules in an afternoon. The reaction is easy to execute. It requires no more than a test tube, a few simple reagents, and a source of heat."What is RT PCR?RT-PCR (Reverse transcriptase-polymerase chain reaction) is a highly sensitive

31、 technique for the detection and quantitation of mRNA (messenger RNA). The technique consists of two parts:· The synthesis of cDNA (complementary DNA) from RNA by reverse transcription (RT) and· The amplification of a specific cDNA by the polymerase chain reaction (PCR).RT-PCR has been use

32、d to measure viral load with HIV and may also be used with other RNA viruses such as measles and mumps.PCRIntroductionPCR (Polymerase Chain Reaction) is a revolutionary method developed by Kary Mullis in the 1980s. PCR is based on using the ability of DNA polymerase to synthesize new strand of DNA c

33、omplementary to the offered template strand. Because DNA polymerase can add a nucleotide only onto a preexisting 3'-OH group, it needs a primer to which it can add the first nucleotide. This requirement makes it possible to delineate a specific region of template sequence that the researcher wan

34、ts to amplify. At the end of the PCR reaction, the specific sequence will be accumulated in billions of copies (amplicons).How It WorksThe PCR reaction requires the following components:DNA template - the sample DNA that contains the target sequence. At the beginning of the reaction, high temperatur

35、e is applied to the original double-stranded DNA molecule to separate the strands from each other.DNA polymerase - a type of enzyme that synthesizes new strands of DNA complementary to the target sequence. The first and most commonly used of these enzymes is Taq DNA polymerase (from Thermis aquaticu

36、s), whereas Pfu DNA polymerase (from Pyrococcus furiosus) is used widely because of its higher fidelity when copying DNA. Although these enzymes are subtly different, they both have two capabilities that make them suitable for PCR: 1) they can generate new strands of DNA using a DNA template and primers, and 2) they are heat resistant.Primers - short pieces of single-stranded DN