I am cloning a whole cDNA of a particular gene and I have designed forward primer at start of 1st exon. The region downstream of forward primer is extremely high GC rich (around 85-89%). Because of this region my FP couldn't amplify even if suppose it has annealed to the DNA correctly. I have tried different conditions like changing denature/melting temperatures, increasing time, and using different polymerases viz., Prime star Max/ Prime star GXL. However none of them seems to be working.
Any leads from those who have/had encountered similar problems in cloning a very high GC-rich regions are appreciated. Thank you!
try adding betaine to a final concentration of 1Molar in the pcr mix. Also try adding both dmso 6% and betaine ( final concentration of 1 molar) both of which are cheap reagents. If neither of these methods works well then you may have to look at using base analogues like 2 deoxycytidine in partial replacement of C to break the secondary structure problems. Also keep the temperature of the pcr as hot as possible at all times so possibly design longer primers to get a high annealing temperature
Hi,
you can try overlap PCR or adjust your protocol with DMSO. Preprint PCR procedures to amplify GC-rich DNA sequences of Mycobacterium bovis
try adding betaine to a final concentration of 1Molar in the pcr mix. Also try adding both dmso 6% and betaine ( final concentration of 1 molar) both of which are cheap reagents. If neither of these methods works well then you may have to look at using base analogues like 2 deoxycytidine in partial replacement of C to break the secondary structure problems. Also keep the temperature of the pcr as hot as possible at all times so possibly design longer primers to get a high annealing temperature
Additionally you could try amplifying in 2 sections of overlapping template....a shorter amplimer of the high gc region (to help the melting part of the pcr) and also amplifying the rest of the template then as the amplimers overlap you can mix and melt and reanneal to get some full length overlapping product that can be made double stranded with pcr reagents
Dear P. Rutland and S. Mukta,
Thank you very much for your valuable answers. I do work on them.
Kind regards,
Harsha Somashekar
The Phusion DNA polymerase is pretty suitable for amplifying difficult (GC-rich) or long amplicons. If your lab has the enzyme, you can try it using its GC buffer with/out DMSO.
Dear Harsha Somashekar
We also had a similar issue with one of our cloning. However, we sorted that out using 10% DMSO and Phusion Taq Polymerase (As Abeeb mentioned above). Phusion TAq is a great enzyme for such difficult PCR reactions.
Alternatively, using two short amplicons followed by a Overlap extension (also using DMSO and GC Buffer Mix) could sort it out.
Regards
Rudra
Dear Rudrarup Bhattacharjee,
Thank you very much. I will definitely try them.
Regards,
Harsha
Hi Harsha Somashekar
According to my experience, using the Phusion enzyme (Phusion® High-Fidelity DNA Polymerase) is a great idea. GC buffer (improves reaction performance) and DMSO have been included in this product for targets with GC-rich sequences or secondary structures. Also, it is important to know that a high concentration of DMSO decreases the primer Tm, so the annealing temperature should be lowered.
Good luck
Hi Shirin Parizad
Thanks so much for your suggestions, I will definitely try it out.
Kind regards
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