Abstract
DNA polymerases with proofreading activity are important for accurate amplification of target DNA. Despite numerous efforts have been made to improve the proofreading DNA polymerases, they are more susceptible to be failed in PCR than non-proofreading DNA polymerases. Here we showed that proofreading DNA polymerases can be inhibited by certain primers. Further analysis showed that G-rich sequences such as GGGGG and GGGGHGG can cause PCR failure using proofreading DNA polymerases but not Taq DNA polymerase. The inhibitory effect of these G-rich sequences is caused by G-quadruplex and is dose dependent. G-rich inhibitory sequence-containing primers can be used in PCR at a lower concentration to amplify its target DNA fragment.
Highlights
As secondary structure of the G-rich oligonucleotides are normally disrupted during Polymerase chain reaction (PCR), and the inhibitory effect of G-rich sequences could be reduced by adding its complementary oligonucleotide (Fig. 4), one may concluded that the inhibition of proofreading DNA polymerases is caused by single-stranded G-rich sequences
The use of proofreading DNA polymerases to amplify target DNA fragments is highly demanded for cloning of targets without undesired mutations
We showed that oligonucleotides containing G-rich sequences such as GGGGG and GGGGHGG can inhibit proofreading DNA polymerases in a dose-dependent manner
Summary
Primers inhibit proofreading DNA polymerases during PCR. Our results showed that the primers Stat3R and GfapR showed an inhibitory effect to PS (Fig. 2B), indicating that the primers inhibit the DNA polymerase directly rather than inhibit the processivity-enhancing factor. LATaq was not inhibited by the primers Stat3R and GfapR, whereas all the tested proofreading DNA polymerases such as Phusion, Q5, Cobuddy, PS, PSGXL and PfuFly were substantially inhibited by the primers Stat3R and GfapR (Fig. 2C).
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