High Fidelity PCR

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First of all some words about the reagent selection for high fidelity PCR.

High fidelity PCR is frequently used for cloning and mutagenesis. However standart Taq polymerase is not sutable for this reaction, since it simply makes to many mistakes during amplification. There are many various high fidelity PCR enzymes available on the market. In addition many companies try to sell you the "latest development" the "best enzyme ever" and so on products. It may be really hard to choose the right enzyme. In our lab we used Expand High Fidelity PCR system from Roche for years. But once I decided to forget for a moment "Never change the winning team" principle and decided to try several novel "extreme high fidelity enzymes". The result was much below expectation. First of all none of the products performed better than the Roche one. But this was not enough. One enzyme (that was actually the most expensive one) failed even to amplify a relatively simple target! So we decided to return to "Expand High Fidelity System" and stay with it. It works fine for all types of templates (plasmids, cDNA, genomic DNA) and nicely accepts 10% DMSO in the PCR reaction if GC-rich template has to be amplified.

Another issue of high fidelity PCR is the quality of nucleotides. Every sales rep of a company that sells dNTPs will try to sell you high purity (and expensive) dNTPs, since "surely you results are not good enough since your dNTPs are not good enough!" Forget it! For all types of PCRs we run in the lab we use a standart Fermentas product. It is cheap and does its job perfectly.

Now to the reaction itself.

We use the protocol proposed by the company.

The reaction mix contains

Buffer (5x) 10 µl
dNTPs (10mM) 1 µl
forward pimer (4µM) 5 µl
reverse primer (4µM) 5 µl
Enzyme (5 U/µl)   0.5 µl
Template variable
H2O  to 50 µl

As a template we usually take 1-50 ng plasmid DNA. It it important to note, that usage of large amounts of templates reduces the quality of the reaction. Usually we do not perform more than 25 cycles. Elongation step is programmed at 72°C considering synthesis speed to be 1 kb/min, but not less than 45 seconds.

If you are trying to amplify a GC-rich region, you have to optimise the reaction adding 1, 3 and 10% DMSO. With 10% DMSO I managed to amplify fragments with as much as 80% GC.

Although the prolymerase works really with high fidelity we usually sequence all clones obtained this way. With fragments up to 2 kb we usually have 1 or 2 mutated clones (usually not more than 1 mutation) out of 10.


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