Using PCR to create overhangs - (Sep/22/2010 )

I was hoping that someone could shed some light on a couple of questions regarding creating overhangs using PCR (i.e. inserting bps at the beginning or end of a target sequence):


1) When calculating what melting temperature to use for the PCR reaction, how should one take into account the overhang (since the first amplification will not use the overhang while all the subsequent amplifications will)?

2) In general, what is the limit of how many base pairs one could create with an overhang and still have a reasonable success rate with PCR reactions (e.g. can a 10 bp overhang work?)

Thanks!
-Albert

I assume that u mean creating overhang is introducing RE sites in both the primers 5'...if tat is the case i normally use NCBI primer blast to design my primers, i design primers for 60(+/-2) melting temp, then i introduce the RE sites, for most of the enzymes 9 bases (6 RE+3 extra) minimum u have to introduce to create a Restriction site...for that i jus set up the Annealing around 60, it mostly works fine without non-specific bands. hope this help...good luck...

Gnana...

GNANA on Wed Sep 22 21:24:54 2010 said:

Thanks! I'll try that out.
-Albert

American3pt14 on Thu Sep 23 15:08:09 2010 said:

In my experience it depends a lot on the length of the overhang what result you will get. I've done PCRs with overhangs (generated by another PCR) over 1000Bp. It has not reasonable effect on the melting temperature that you should care about, it is just denatured the same as the DNA double strand of the template when it has been fused to the old template.
What you definitely should do though if your overhangs are longer than just REN site and a bit extra for digestion: test the hairpin and self-annealing capacity of the 3' end with Netprimer. You need to register, it is an online tool, but it is for free. If the 3' end of your primer is self-annealing too much with the overhang you are not going to have bands or very little PCR efficiency. The 5' end is overhang and thus not important of course.
In case your primer is overlapping only a bit with the template prior to the fusing (like the melting temperature is only 54C in the beginning as for whatever reason it can't be longer) I make something like a step-up PCR:
1. long denature/enzyme activation
2. short denature
3. anneal at 54C
4. extension
5. repeat 2-4 4x
6. short denature
7. anneal 60C
8. extension
9. repeat 6-8 25x
10. remainder extension