Update: Thank you everyone--what I should have included was that the fragment forms tetra-G structures. It's a 600 bp long sequence with 80 repeat of GGGG (with 4 non-G base pairs in between). Does anyone have any way of amplifying this
I'm trying to amplify two oligos comprised of repeat sequences that are each about 72% GC rich. One fragment is about 550 bp and a second nearly 3kb long. I was wondering if anyone had any advice/success with a protocol for such a GC rich amplicon.
Try with AmpliTaq Gold 360 PCR Master Mix (Cat # 4398881) by Thermo Scientific, is very effective for high GC rich PCR. I am tested it in plant sample PCR for 66% GC.
As mentioned by Shuvobrata, suppliers of DNA polymerase usually provide alternative mixes/buffers to facilitate the amplificationi of GC-rich DNA, sometimes at the cost of a somewhat decreased fidelilty. However, if amplification still fails after several trials, I suggest to order a synthetic fragment from a gene synthesis company. The cost does not exceed a few hundred of $, even for your large fragment, and if you plan to use it for expressing a recombinant protein, you can ask for optimization of the codons.
use primers with a very high annealing temperature to minimise strand reassociation and run the pcr in a final concentration of 1molar betaine to weaken the effect of the gc triple hydrogen bonding. make the annealing time very short again to minimise reassociation
Dear Danielle,
I think it will be difficult to amplify such big amplicons with high specifity.
I would try to amplify your amplicons with other primer target sequences (with 50% to 60% GC content) before and after your target sequence.
Best regards,
Thomas
Your amplicon size is too large still you can go through this protocols
https://academic.oup.com/nar/article/25/19/3957/2549226
Article DMSO and Betaine Greatly Improve Amplification of GC-Rich Co...
Hope it will help you.
You can use the Phusion® High-Fidelity DNA Polymerase with the buffer for amplicons with high GC content. Additionally, you can add DMSO at 0.5-5% to the reaction mix in order to decrease the Tm or add deionized formamide instead.
Good luck,
JA
I suggest using 5% (v/v) DMSO. You may reduce the concentration even upto 1% through optimization. It should work.
Best of luck.
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