Primers.

Gene Studio allows to make primers. To make primers select the sequence to amplify and use menu Analysis>Make primers. It will launch the primer wizard, which has three steps. On the first step user should select amplification sequence and primer location. By default position of amplification sequence are the sane as begin and end of selected region. If no region were selected fields "From" and "To" will contain the cursor position. Programs can look for primers inside this region (it is only one primer pair), or outside it. The last approach can be used to find the primers with the most optimal parameters. The "flank length" defines the possible length of flank from both side. To search primers outside region put the mark next to "Outside region". If the flank can be allowed from only one side of a sequence put mark across "Forward inside, reverse outside" (5' flank is not allowed) and "Forward outside, reverse inside" (3' flank is not allowed). Note that when use "Outside region" option the number of primer pairs is 2*"Allowed flank length+1"2. After pressing "Next>>" button the length of forward and reverse primers should be determined. And on the last step melting temperature constraints should be entered. Currently three types of constraints are available: lower Tm border, higher Tm border and difference of Tm between forward and reverse primer. By default Tm constraints are switched off. To activate Tm lower border constraint check "Tm not lower then", check "Tm not higher then" to activate higher border and to activate constraint on difference in primer pair Tm activate "Tm difference not higher then". It is possible to move through wizard screens bu pressing "<<Back" and "Next>>" buttons. On the third step after all settings are entered press "Finish". The window with primer table will shown up. The primer pare information in this window is ordered by increase of Tm difference between two primers and by ability of one of primer to form secondary structure. Primers which can form secondary structure are placed at the bottom of the list. Primers are proposed to have secondary structure if they contain three complementary G/C pairs separated by at least seven nucleotides. The information about primer can be copied or saved to the CSV file using "Save..." button. Tm is determined by empirical rule Tm="Number of G/C"*4+"Number of A/T"*2. Primers are shown from 5' to 3'.



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