I need to amplify a 2kb gene from the genome. The details of the primers are as follows:
FP - GC - 70%, Tm - 76o C
RP - GC - 73%, Tm - 77o C
do the gradient again but add betaine at a final concentration of 1 Molar to all tubes. Betaine destroys the hydrogen bonding beteen the strands so the template melts esier. If your templte is GC rich then it will anneal very quickly and easily so the pcr will not work unless you use something like betaine to keep the strands melted while the primer sticks and extends. It is very cheap
You may also need to check the polymerase you are using. I experienced similar problem but when I change my polymerase I got my band. With 2kb amplification I suggest you try High fidelity enzymes.
You can also try with increasing the concentration of Mg and NH4 ions or use foramide for the high GC rich Template.
1. Sarkar, G., Kapelner, S., and Sommer, S. S. (1990) Formamide can dramatically improve the specificity of PCR. Nucl. Acids Res. 18, 7465
You can also use GC-Rich PCR system of Roche or Phusion High Fidelity (Thermo Fisher) with its corresponding GC buffer. The first one works very well, even with the trinucleotide GCC of fragile - X syndrome.
Good luck!
Instead of betaine, you could also use DMSO. It usually comes with the polymerases. But you need to lower your Tm a bit, so running the gradient again would be a good idea.
Take a look at my post in this thread which includes a link to a paper for primer design and PCR conditions which I found very helpful:
https://www.researchgate.net/post/How_can_I_sequence_human_COQ2_DNA_exon_1/1
High GC content of the gene generates complication during primer designing like mismatch and high annealing temperature, self-dimer formation, and secondary structure. Sometimes, amplification of gene is not routinely achieved by normal PCR techniques. The most prominent problem associated is hairpin loop, which directly interferes during annealing of primers on difficult DNA template that leads to no amplification. Different strategies have been proposed to sort out this problem. Use of DMSO and glycerol was reported to reduce the annealing temperature and denaturation temperature, increase the chances of breakage of secondary structure, and increase the efficiency of amplification.
1- Winship PR. An improved method for directly sequencing PCR amplified material using dimethyl sulphoxide. Nucleic Acids Research. 1989;17(3):1266–1269
2- Weissensteiner T, Lanchbury JS. Strategy for controlling preferential amplification and avoiding false negatives in PCR typing. BioTechniques. 1996;21(6):1102–1108.
3-Varadaraj K, Skinner DM. Denaturants or cosolvents improve the specificity of PCR amplification of a G + C-rich DNA using genetically engineered DNA polymerases. Gene. 1994;140(1):1–5
4- Pomp D, Medrano JF. Organic solvents as facilitators of polymerase chain reaction. BioTechniques. 1991;10(1):58–59
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