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cloning primers, TM, and specificity - (Feb/20/2007 )

I have primers that work very well for cloning cDNA, if I include non specific sequences at the 5' end of some new primers (anywhere from 30-70 more bps) that should not effect the specificy or efficiency of the primers should it?

(I do intend on keeping the same TM of the old 20bp primers for the first 10 cycles or so, then swithing to a higher annealing temperature for the final 25 cycles, or should I keep it low?)

One more question, do I still have to match the TMs of the nonspecific sequences attached to the new primers?

For instance if my old primer set has a TM of 58 degrees, but when I add the nonspecific sequences my new F primer has a TM of 75, and my R primer has a TM of 69, everything should amplify if I make my annealing temperature ~68?

EJ

-Eric J-

um, this is a little confusing but I'll take a stab

of course, adding bases will change the specificity and efficiency. you will be amplifying different parts of the template, if I'm understanding you correctly. are you looking for DNA that is flanking certain conserved regions, or perhaps certain known regions?

anyways, yeah you do need to consider the changed TMs of your primers. could you please explain why you are keeping the same TA for the first few cycles? what exactly are you accomplishing?

-aimikins-

let me rephrase, I don't think I was clear initially.

I am able to clone the cDNA of my gene of initerest with primers P1 and P2 (both with TMs of 59). Now I want to add a kozak sequence and restriction site to the 5' end and a short peptide coding sequence to the 3' end of the gene. To do this I will add those sequences to P1 and P2 respectively to make P3 and P4 respectively (standard practice). The TM for P3 is 69 and the TM for P4 is 75. I will clone the PCR product into pBS.

My PCR program (as suggested here):
98 for 30 seconds
98 for 10 sec
58 for 20 sec
72 for 1 min
steps 2-4 10 times

98 for 10
72 for 1:20
25 times

72 for 5 minutes
4 for ever.

My questions are:

1) will P3 and P4 still bind to and amplify my cDNA of interest at 58 degrees for the first 10 cycles? will there be reduced efficiency or specificity?

2) When I am designing primers P3 and P4, do I still have to match the TMs of the total primers? (even though the TMs of the gene specific portions of the primers are the same).

Does that clarify or confuse you even more.

Thanks,
Eric J.

p.s. I just realized I asked a similar question six months ago...I'm still not quite clear though

-Eric J-

I think you should do it as you have suggested. Of course you can expect less specificity because you have added "non-specific" bases to the end, but initially these bases cannot be used to determine Tm because they are not present in the target sequence. Anneal at your usual Tm for a few rounds and then increase Tm because the full sequence of the primer is in the products from the first few rounds.
I think I would try that first, then if there are problems you may adjust... You should still re-BLAST your new primer sequences because you will have different mismatches with the new primer set... make sure there isn't anything obviously bad with the new primer sequences and go for it!

-beccaf22-

QUOTE (Eric J @ Feb 21 2007, 09:42 AM)
My questions are:

1) will P3 and P4 still bind to and amplify my cDNA of interest at 58 degrees for the first 10 cycles? will there be reduced efficiency or specificity?

2) When I am designing primers P3 and P4, do I still have to match the TMs of the total primers? (even though the TMs of the gene specific portions of the primers are the same).

I disagree with beccaf. The PCR with P3 and P4 will work equally as well as your PCR with P1 and P2 . The extra 5` sequence on each primer may bind to secondary sites, but more importantly the original 3` portion of your primers will not bind to secondary sites because they didn't in the original PCR. The 3` portion of the primers are what your target sequence is extended/amplified from and they do not bind to secondary sites, so therefore your second PCR will work just as well provided you use the same conditions as your original PCR.

To answer your specific questions
QUOTE (Eric J @ Feb 21 2007, 09:42 AM)
1) will P3 and P4 still bind to and amplify my cDNA of interest at 58 degrees for the first 10 cycles?

Yes, but if you're going to change the annealing temperature after 10 cycles don't expect the result to be the same as your original PCR because you're changing the conditions. Keep the conditions the same and the same result will appear.

QUOTE (Eric J @ Feb 21 2007, 09:42 AM)
Will there be reduced efficiency or specificity?

Maybe very slightly just based on the fact that they are slightly different, although practically identical primers given their identical sequence at the 3` ends. It's all about the 3` end in PCR and here it is identical.

QUOTE (Eric J @ Feb 21 2007, 09:42 AM)
2) When I am designing primers P3 and P4, do I still have to match the TMs of the total primers? (even though the TMs of the gene specific portions of the primers are the same).

No. Two things here. 1) The Tm is always measured using the 3` portion of the primers that matches the target exactly because that's the sequence the primers have to anneal to in the first cycle in order for the amplification to begin. If you start considering the Tm of the longer primers and use that temperature, no sequence can be amplified from the original target because the annealing temperature will be too high for the primers to anneal to that sequence. You could use the higher temperature after 10 cycles I suppose, that may improve specificity, but it will also reduce yield and given that specificity is not a problem here there's no advantage to doing that. 2) Matching the Tm of each primer is not absolutely essential. It just helps reduce the chance of the primer with the higher Tm annealing to secondary targets (because the annealing temperature will be much lower than the Tm of the higher Tm primer and that reduces the specificity of that primer for its target). If the specificity of the primers or the annealing temperature of each primer is high enough this is not a major issue.

-killerkoz17-