long primers PCR - (Jan/10/2013 )

Hi

For a while I've been trying to amplify different regions on human chromosome 21 q arm. I'm using long primers (60-90 bp) with high theoretical Tm between 71 and 72 deg C.



I've tested several different annealing temperatures in the range from 65-69 deg C. With one primer pair I get a moderately strong PCR product at 69 deg C.The last attempt I made was a two stage PCR with annealing temperature combinations

69 C-10 cycles/70 C-15 cycles,

69 C-10 cycles/71 C -15 cycles,
69 C-10 cycles/72 C -15 cycles,
69 C-10 cycles/74 C -15 cycles

and it didn't work.

I'm using HiFi polymerase by Kapa Biosystems with GC buffer. The initial denaturation is 98 deg for 5 min. Denaturarion in each cycle is 98 deg for 20 sec. The manufacturer suggests using two stage PCR when long primer pairs are used.


Anyone has any advice regarding the amplification?

Thanks

Often long primers are designed having a relatively short (18-24 bp) region homologous to the target region of DNA, and a long 5' region having nothing to do with the target. In these cases, the primer melting temperature is very misleading, since it will be calculated on the entire primer, rather than on the portion binding to the target. I'd recommend you retry your PCR with a much lower annealing temperature, 55, for example.

The recommended annealing temp for KAPA HiFi is from 60-78C, that's quite high. I would use a different polymerase if I were you, considering you are already using long primers.

Thank you both.

Except for the 12 bases at the 5 end side where I have my restriction site, the rest of the primers anneal to the target sequence.

I'm trying one more round with the Kapa HiFi strictly following their technical datasheet info. For the long primers which have the Ta above 68 deg they recommend two stage PCR setup but in my case it doesn't work.
If this last attempt doesn't work I'll switch to Pwo and see what happens.

What is the reason for using such long primers? Too long primers are know to actually bind only with the distal 3' part where required, thus not actually adding much to the primer specificity. Also the real annealing temperature would be substantialy lower than calculated.
But from the fact you only got PCR product with the higher temperatures I assume maybe the main problem is secondary structures on your (very long) primers. But since you already use GC buffer with probably addition of DMSO or some other secondary structures-relaxing reagent, it's probably useless to try add DMSO..