I have a gene that I need synthesized that is about 4150 bp long. Getting it synthesized completely in one piece will be expensive and time-consuming. I've used overlap PCR before to make ~1000 bp products, but I'm wondering if I can instead use overlap PCR to connect the two halves of my gene? (~2 pieces each at 2000 bp for ~4000 bp total) . Synthesizing fragments ~2000 bp is much cheaper and faster.
Thanks in advance!
Andrew
Performing the overlap pcr as you suggests should be no principal problem in my opinion. The only thing is of course that the risk of random mutations go up with pcr length, but if you use a proof-reading polymerase this should be fine. You could also look into cloning using some of the more recent assembly-based methods, for instance Gibson assembly, instead of traditional, ligation-based cloning. Using these methods you can directly assemble your vector + 2 gene fragments in one step without needing to do the overlap extension first.
I have also used overlap extension PCR for smaller products. As long as the overlap region is unique and has the properties desired of a primer, it should work.
Hi Andrew, I have used a hi-fi (High Fidelity) PCR premix which was quick, reasonably priced and only one reaction (4 hours) to amplify my gene of interest of 3987bp. Anyway, best wishes for your overlap extension PCR idea.
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