PCR的应用.pptVIP

Cloing The Unknow Sequence by PCR five categories be used in cloning PCR after intramolecular circularization Interspersed repetitive sequence PCR (IRSPCR) Ligation-anchored PCR (LA-PCR) Arbitrarily primed PCR (AP-PCR) Reverse transcription PCR (RT-PCR) PCR after intramolecular circularization A PCR technique, which is used to amplify an unknown DNA region adjacent to an integrated sequence after its intramolecular circularization, is called IPCR (inverse or inverted or inside-out PCR). Inverse PCR Partial inverse PCR (PI-PCR) Partial inverse PCR (PI-PCR) employs the genomic DNA partially digested by using 4-base recognition restriction enzyme (such as Sau3A1). After self ligation ,the circular DNA fragments are used as templates for IPCR . This is based on the preference of PCR for amplifying relatively smaller fragments. A DNA fragment that is less than 1 kb facilitates amplification via self-ligation and the IPCR process . PI-PCR Long-distance IPCR Designated long-range IPCR (LR-iPCR) or longinverse PCR (LI-PCR) , Relatively large size ：up to 10 kb Thermostable polymerase. advantage Amplify the flanking unknown sequence by using two known specific primers on the integrant limit The noncircularized, intermolecular ligation, and free viral or transposon DNA fragment may interfere with the PCR. Intermolecular ligation generate multiple PCR products correct:(decrease the concentration of DNA and this results in a large volume for ligation) IRS-PCR-Based Techniques The principle of IRS-PCR (interspersed repetitive sequence PCR) is based on the fact that the IRS elements are interspersed in the human genome. One ISP to the known integrant sequence and the other primer specific to the known cellular IRS. IRSs High copy number in most multicellular organisms Novel Alu-PCR Novel Alu-PCR (novel Alu element-mediated PCR) is an IRS-PCR, which uses a primer to Alu element and ISP for the PCR amplification. Alu elements :SINEs, 3–6 kb(human), other B1-PCR:has b