[理学]第四组分子PPT PCR.ppt

Let’s Talk PCR ！ Members：胡忠生 卢芳芳 吴晋 傅瑶 魏亚林 荣周易 秦昕 Contents Introduction 1 Brief History 2 Techniques and Applications 3 Introduction PCR is a powerful method to amplify specific sequences of DNA from a large complex mixture of DNA. For example, you can design PCR primers to amplify a single locus from an entire genome. From a single template molecule, you can produce over 1 billion copies of the PCR product very quickly. However, the capacity to amplify over one billion fold also increases the possibility of amplifying the wrong DNA sequence over one billion times. The specificity of PCR is determined by the specificity of the PCR primers. For example, if your primers bind to more than one locus (e.g. paralog or common domain), then more than one segment of DNA will be amplified. To control for these possibilities, investigators often employ nested primers to ensure specificity. The basic principle of PCR PCR reaction components： 1.DNA template； 2.Primer； 3.Four dNTPs；4.DNA polymerase； 5.Reaction buffer、Mg2+ etc. PCR reaction procedure The single stranded primer and template bond together （55℃，30s） Denaturation Annealing Extension The double strand melts open to single stranded DNA（94℃，30s） The bases are coupled to the primer on the 3’ side （70～72℃ 30～60s） The three steps constitute a cycle and the production of every cycle becomes the template of the next cycle. Target DNA increases quite a lot after 30 or 40 cycles. PCR reaction procedure Brief History 1971 Khorana 1985 Kary Mullis Early 1988 Keohanog Saiki 1998 21世纪 ？？？ PCR Techniques PCR Real- time quantitative PCR Nested PCR In Situ PCR Reverse PCR Asymmetric PCR Reverse transcription PCR Nested PCR Nested PCR means that two pairs of PCR primers were used for a single locus. The first pair amplified the locus as seen in any PCR experiment. The second pair of bind within the first PCR product and produce a second PCR product that will be shorter than the first one. The logic behind this strategy is