Hi everybody, I want to know the critical steps in designing primers, means what will suitable Tm value, GC content, false priming, dimer, cross dimer, hairpin, rating, length of primer, product size? and please tell me what is best annealing temperature for such kind of primer Tm? and how to find Tm value for sense and anti sense primers together?thanks, reply quickly its very urgent to know.
Dear Bushra
I have come to this discussion late
I have written about and designed primers many times; In terms of optimal design see a plethora of previous correspondence of mine on this subject and also doc attached
In essence:
1. Primers should be 18-22bp in length; 40-50% GC. This equates to a Tm of 60-65C which is optimal
2. Pay attention to the last 5bp of the primer in particular: Try and ensure that the last 5 bases have no more than3 x G/C residues. Moreover 1-2 G/C residues at the terminus of the primer are acceptable (indeed current thinking prescribes that 2 x GC lead to more stable priming and efficient PCR) but not all 3 residues (as this predisposes to primer dimers and mis priming)
3. To compute the Tm of a primer use the following program:
https://www.idtdna.com/calc/analyser
Make sure you fill in the fields relating to primer concentration mg and dNTPs as these will affect the actual Tm in the context of your reaction
4. Make sure having computed Tm that your forward and reverse primer have a Tm which differs by no more than 2C
5. Now check for primer (heterodimers) dimers between forward and reverse primers using the above program:
Anything with a delta G value > -10KCal/Mol is likely theoretically to be stable and contribute to real steady state primer dimers. These tend to equate to binding at either end of the primer through > 3 x G/C residues. Hence the reason for avoiding multiple G and C residues at the termini of either primers
Ignore predicted structures with delta G values < -10kCal/Mol as these will not be stable: Such structures tend to be theoretical binding in the middle of the primer; Via non contiguous bases and sometimes through A/T residues/ In reality these structures do not exist in any stable or meaningful way so simply ignore
6. Now look for intra primer annealing - either a self primer binding between the same species of primer or hair pin loops. I tend to use the following program for this:
http://biotools.nubic.northwestern.edu/OligoCalc.html
7. Finally BLAST search the primers to make sure they only bind 100% to your target of interest; You can perform that function directly from the aforementioned Oligo properties calculator
It is worth noting that if you select your target sequence and then design primers using primer 3 or primer BLAST the above parameters are built into primer selection:
http://primer3plus.com/
http://www.ncbi.nlm.nih.gov/tools/primer-blast/
8. Finally in terms of annealing temp and PCR Tm-2C tends to be optimal but if that isn't quite optimal try Tm-3C to Tm+2C in 1C intervals: With some of the newer polymerases like Phusion an annealing temp corresponding to the Tm will work very efficiently; Unlike WT Taq Polymersase where generally speaking the Tm -2 C = Ta rule tends to apply
Hope this helps !!
Dear Bushra
I have come to this discussion late
I have written about and designed primers many times; In terms of optimal design see a plethora of previous correspondence of mine on this subject and also doc attached
In essence:
1. Primers should be 18-22bp in length; 40-50% GC. This equates to a Tm of 60-65C which is optimal
2. Pay attention to the last 5bp of the primer in particular: Try and ensure that the last 5 bases have no more than3 x G/C residues. Moreover 1-2 G/C residues at the terminus of the primer are acceptable (indeed current thinking prescribes that 2 x GC lead to more stable priming and efficient PCR) but not all 3 residues (as this predisposes to primer dimers and mis priming)
3. To compute the Tm of a primer use the following program:
https://www.idtdna.com/calc/analyser
Make sure you fill in the fields relating to primer concentration mg and dNTPs as these will affect the actual Tm in the context of your reaction
4. Make sure having computed Tm that your forward and reverse primer have a Tm which differs by no more than 2C
5. Now check for primer (heterodimers) dimers between forward and reverse primers using the above program:
Anything with a delta G value > -10KCal/Mol is likely theoretically to be stable and contribute to real steady state primer dimers. These tend to equate to binding at either end of the primer through > 3 x G/C residues. Hence the reason for avoiding multiple G and C residues at the termini of either primers
Ignore predicted structures with delta G values < -10kCal/Mol as these will not be stable: Such structures tend to be theoretical binding in the middle of the primer; Via non contiguous bases and sometimes through A/T residues/ In reality these structures do not exist in any stable or meaningful way so simply ignore
6. Now look for intra primer annealing - either a self primer binding between the same species of primer or hair pin loops. I tend to use the following program for this:
http://biotools.nubic.northwestern.edu/OligoCalc.html
7. Finally BLAST search the primers to make sure they only bind 100% to your target of interest; You can perform that function directly from the aforementioned Oligo properties calculator
It is worth noting that if you select your target sequence and then design primers using primer 3 or primer BLAST the above parameters are built into primer selection:
http://primer3plus.com/
http://www.ncbi.nlm.nih.gov/tools/primer-blast/
8. Finally in terms of annealing temp and PCR Tm-2C tends to be optimal but if that isn't quite optimal try Tm-3C to Tm+2C in 1C intervals: With some of the newer polymerases like Phusion an annealing temp corresponding to the Tm will work very efficiently; Unlike WT Taq Polymersase where generally speaking the Tm -2 C = Ta rule tends to apply
Hope this helps !!
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