I want to design a primer where I want to insert an overhang of size 32bp whereas the size of annealing sequence is 32bp( GC=56%). Please tell me your views.
When doing recombinant PCR on AT-rich sequences, I've had success with as much as a 35-base overhang with a 35-base annealing sequence at the 3' end. As long as your annealing sequence is a perfect match and has no hairpins, it should work fine. When you order a primer that is that long, be sure to get it purified! Purification at least gets rid of primers that are actually missing bases. The longer the primer, the higher the probability of a DNA-synthesis error.
Ideally, at least half of your primer should encompass your existing sequence (although I have gone down to as little as a third and had success), to ensure that the 3’ end of the primer can bind to your target sequence. The rest of your primer can be your overhang. You want to aim for primers about 25 basepairs in length, but it depends on how big your desired overhang is (my largest primers were over 100 basepairs).
http://bitesizebio.com/20786/overhang-pcr/
It seems too big. There is no strict rule for designing this types of primer. Bigger size primer will end up in more secondary structure and leading to no amplification. In a 32 nucleotide primer you can easily add 10-15 additional nucleotides.
When doing recombinant PCR on AT-rich sequences, I've had success with as much as a 35-base overhang with a 35-base annealing sequence at the 3' end. As long as your annealing sequence is a perfect match and has no hairpins, it should work fine. When you order a primer that is that long, be sure to get it purified! Purification at least gets rid of primers that are actually missing bases. The longer the primer, the higher the probability of a DNA-synthesis error.
Thank you so much to everyone for giving me your valuable suggestions. I tried Touchdown PCR and I got the desired result.
Dear Avinash,
I am having the same problem. I've designed primers 45 bp long with overhangs but I am struggling with DNA amplification. Ive tried:
- normal PCR procedure
- touchdown PCR
- Tm gradient
- concentration gradient (primer and DNA template)
- changed Taq poly
I have even started again with fresh solutions but I am still not getting any amplification at all.
could you guys help me please.
Hi Abdulrahman,
How big are your overhangs in terms of the bp of your primer? Try ordering the primers without overhangs and seeing if you get the correct sized product, then purify this and use your primers with overhangs to amplify up the desired DNA sequence. This I guess is a type of nested PCR. Also, use KOD hotstart master mix for your polymerase, it works very well for me, better than Phusion.
Good luck!
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