I want to know that during Design Primers to check the effect of Protein Bait on the Gene Regulations of of Bactrocera dorsalis which key points should kept in mind regarding GC contents, Anhiling Temp.? and why some scientist not suggest good primer if AAATTT or any of 3 base pairs come consecutively in primer.
Dear Atif
Find attached an SOP in the form of a word doc that I use to teach students primer design
In essence and see my word document plus accompanying pdf for details you need to:
1. Make sure your primer is between 18-30bp and has a GC content of ~ 50%: This will result in a melting temp of between 55 and 65C. Most applications like PCR and qPCR require primers with annealing temp of ~ 50C to 65C so primers of this length and GC content will be compatible with these annealing temp
2. Make sure your forward and reverse primers have Tms in this range +/-2C; Ideally the same Tm: If you select primers with programs like primer blast/primer quest or primer 3 they will generally give you primers within these limits
3. The annealing temp of your PCR/qPCR is generally Tm-2C. Better still take your primer and perform a PCR gradient from Tm+1C to Tm-5C in 1C temp intervals: Pick the annealing temp which gives a strong specific band; That is the PCR is both specific and efficient. Generally, this is the Tm itself to Tm-2C but can be as low as Tm-5C. If you primers do not work @ less than Tm-5C they are probably null primers and will not work. Thus, re design
4. As per my document calculate the Tm using programs like IDT and Oligo calculator, viz
http://www.idtdna.com/calc/analyser
5. Also, as elaborated in my word doc screen the primers for hair pin loops and self annealing using Oligo calculator:
http://www.basic.northwestern.edu/biotools/oligocalc.html
and then heterodimers between forward and reverse primers, leading potentially to primer dimers, using the IDT oligo tool:
http://www.idtdna.com/calc/analyser
Generally speaking - and as detailed in my doc - never start or end primers with more than 3 consecutive G/C residues as this can lead to terminal primer annealing and stable primer dimers. The algorithm for predicting primer dimers refers to thermodynamic stability by quantifying in terms of free energy and anything with a delta G value > -10kcal/mol is likely to be stable and will thus exist in real time in a steady ate manner and therefore bevisible on your gel and undermine the efficiency of your PCR. Putative structures with delta G values < -10kCal/mol are unlikely to be stable and can thus be ignored
6. Apart from ensuring the mid point of your primer is GC rich - your so called GC clamp which anchors your primer to its target - pay attention to the last 5 bases; In particular, try not to include more than 3 G/C residues in the last 5 bases and NEVER end your primer in 3 consecutive G/C residues and as you mention something like TTT (or indeed AAA or combinations of A & Ts): Primers ending in sequences like GCC; GGC; or CCC are more likely to bind non specifically and in addition form primer dimers. Endings like GG; GC; CC; GT; CT; GA and CA are more optimal
7. Finally, having chosen primers 18-22bp (on average) with ~50% GC content and thus predicted Tms of ~ 55C to 65C and having demonstrated that they are not predicted to form primer dimers or self annealing, check specificity of such primers by Primer blast. Do this using the oligo calculator program mentioned or by going directly to NCBI:
http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome
Make sure your primers only bind to your GOI with 100% complementarity
With reference to primer design, you can alternatively submit your GOI sequence to programs like primer 3 or blast and they will design primer pairs for with the above factored in:
http://primer3plus.com/
http://www.ncbi.nlm.nih.gov/tools/primer-blast/
The principal advantage of primer blast is that it is part of NCBI and therefore imports iit gene sequences automatically. Furthermore, if you are making primers to RNA transcripts i.e. from cDNA you can specifically target these transcripts by specifying exon-exon boundary primers which thus will not bind to gDNA; Only your derived transcript
Dear Atif
Find attached an SOP in the form of a word doc that I use to teach students primer design
In essence and see my word document plus accompanying pdf for details you need to:
1. Make sure your primer is between 18-30bp and has a GC content of ~ 50%: This will result in a melting temp of between 55 and 65C. Most applications like PCR and qPCR require primers with annealing temp of ~ 50C to 65C so primers of this length and GC content will be compatible with these annealing temp
2. Make sure your forward and reverse primers have Tms in this range +/-2C; Ideally the same Tm: If you select primers with programs like primer blast/primer quest or primer 3 they will generally give you primers within these limits
3. The annealing temp of your PCR/qPCR is generally Tm-2C. Better still take your primer and perform a PCR gradient from Tm+1C to Tm-5C in 1C temp intervals: Pick the annealing temp which gives a strong specific band; That is the PCR is both specific and efficient. Generally, this is the Tm itself to Tm-2C but can be as low as Tm-5C. If you primers do not work @ less than Tm-5C they are probably null primers and will not work. Thus, re design
4. As per my document calculate the Tm using programs like IDT and Oligo calculator, viz
http://www.idtdna.com/calc/analyser
5. Also, as elaborated in my word doc screen the primers for hair pin loops and self annealing using Oligo calculator:
http://www.basic.northwestern.edu/biotools/oligocalc.html
and then heterodimers between forward and reverse primers, leading potentially to primer dimers, using the IDT oligo tool:
http://www.idtdna.com/calc/analyser
Generally speaking - and as detailed in my doc - never start or end primers with more than 3 consecutive G/C residues as this can lead to terminal primer annealing and stable primer dimers. The algorithm for predicting primer dimers refers to thermodynamic stability by quantifying in terms of free energy and anything with a delta G value > -10kcal/mol is likely to be stable and will thus exist in real time in a steady ate manner and therefore bevisible on your gel and undermine the efficiency of your PCR. Putative structures with delta G values < -10kCal/mol are unlikely to be stable and can thus be ignored
6. Apart from ensuring the mid point of your primer is GC rich - your so called GC clamp which anchors your primer to its target - pay attention to the last 5 bases; In particular, try not to include more than 3 G/C residues in the last 5 bases and NEVER end your primer in 3 consecutive G/C residues and as you mention something like TTT (or indeed AAA or combinations of A & Ts): Primers ending in sequences like GCC; GGC; or CCC are more likely to bind non specifically and in addition form primer dimers. Endings like GG; GC; CC; GT; CT; GA and CA are more optimal
7. Finally, having chosen primers 18-22bp (on average) with ~50% GC content and thus predicted Tms of ~ 55C to 65C and having demonstrated that they are not predicted to form primer dimers or self annealing, check specificity of such primers by Primer blast. Do this using the oligo calculator program mentioned or by going directly to NCBI:
http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome
Make sure your primers only bind to your GOI with 100% complementarity
With reference to primer design, you can alternatively submit your GOI sequence to programs like primer 3 or blast and they will design primer pairs for with the above factored in:
http://primer3plus.com/
http://www.ncbi.nlm.nih.gov/tools/primer-blast/
The principal advantage of primer blast is that it is part of NCBI and therefore imports iit gene sequences automatically. Furthermore, if you are making primers to RNA transcripts i.e. from cDNA you can specifically target these transcripts by specifying exon-exon boundary primers which thus will not bind to gDNA; Only your derived transcript
1. Know your genome (and potential contaminating genomes).
2. Do not accept primers with lots of unspecific blastn matches (go for < 10 almost perfect hits within the 3' half of the primer in the sequences of the respective species).
3. If GC rich, try going for 72° annealing temp according to Marmur GC4 AT2 -4
4. No bad experience with GGG or CGG at 3' ends.
5. If applicable, all primers may end in a G, however make sure all 4 nucleotides are present ("randomly"). G rich 3' ends in both primers may improve processivity (compared to AT rich ends).
6. Avoid palindromic sequences like AAATTT.
These simple measures lead to good results in most instances. Usually further annealing temperature calculation can be ignored.
Good luck
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