PCR聚合酶链反应英语介绍.docVIP

VirologyThe BasicsTaxonomyLinksHaematologyDNA Down UnderPCR Down UnderHOME Headlines DNA Down Under New 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 creates 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 enzymes ideal operating temperature. The enzymes that are used for the PCR are DNA-dependent DNA polymerases (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. 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 speaking, there are three steps identifed by incubating at different tempratures. the three steps make up a PCR cycle. Double-stranded DNA separation or denaturation (D in Figure 1) Primer annealing to template DNA (A in Figure 1) Primer extension (E in Figure 1) Figure 1.A PCR cycle.The 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 primer can hybridise (TM) are also marked. Denaturation At 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 tempera