PCR引物设计（PCR primer design）.doc

PCR引物设计（PCR primer design） The two Windows will be popped out, the Internal Stability (Delta G) window and the Tm window. In Tm window, click on the lower left corner of the button, would come out to primer location dialog box, enter the primer sequences upstream of the candidate position (Primer5 has given), and the length of the primer can by clicking on the Change - Current oligo length to Change. After positioning, click the Upper button of the Tm window to determine the upstream primer. The same method is used to locate the downstream primers. Click the Lower button to determine the downstream primer. After the primers are identified, you can make full use of the powerful primer analysis functions in the Analyze menu. (2) Analyze the first item for the Key info, click on the Selected primers, will give you the general information of two pairs of primers, including primers of Tm value, this value Oligo is adopted on his neighbor method calculation, than Primer5 primers Tm value slightly tall, in this window also the primer Delta G and the 3 end of Delta g. 3 end of Delta G is too high, will be a mismatch in the site to form a double chain structure and cause DNA polymerization reaction, so the absolute value should be smaller, had better not exceed 9. (3) Analyze the second for Duplex Formation, namely the dimers Formation analysis, can choose the upstream or downstream primers, analysis between upstream primer dimers Formation condition and downstream primer dimers, you can also choose to Upper/Lower, namely between the upstream and downstream primer dimers Formation. Primer dimers are the important factors influencing the abnormal PCR reaction, so should avoid design of primer dimers, at least also want to make the design of primers to form dimers is not stable, namely the Delta G value should be on the low side, generally dont make it more than 4.5 kcal/mol, combined with the base pairs not more than three. In the analysis window of Oligo, the dimer and Delt