Abstract
The proofreading activity of the archaeal family B DNA polymerases enables PCR with high fidelity. However, thermostable proofreading DNA polymerases occasionally failed to amplify target fragment that could be amplified by Taq DNA polymerase. We have previously showed that G-rich sequences, which form G-quadruplex, can bind to and inhibit proofreading DNA polymerases. Here we showed that single-stranded oligonucleotides containing sequences of TT(N)mGCCTC can bind and inhibit archaeal family B DNA polymerases but not Taq DNA polymerase. It is very likely that TT(N)mGCCTC inhibits thermostable DNA polymerases during PCR in a single-stranded form. To the best of our knowledge, this is the first example of DNA sequence that could inhibit DNA polymerase in its single-stranded form.
Highlights
The proofreading activity of the archaeal family B DNA polymerases enables polymerase chain reaction (PCR) with high fidelity
To clone the promoter region of Olig[2] from mouse genomic DNA, we failed to amplify the 2.5 kb fragment using Olig2.5 and OligTSSR as primers and PrimeSTAR GXL DNA polymerase (GXL DNA polymerases (DNAPs))
The results showed that the PCR failure caused by Olig2.5 is specific to GXL, which indicated that PCR failure caused by Olig2.5 is not due to inefficient primer-template annealing
Summary
The proofreading activity of the archaeal family B DNA polymerases enables PCR with high fidelity. We have previously showed that G-rich sequences, which form G-quadruplex, can bind to and inhibit proofreading DNA polymerases. We showed that single-stranded oligonucleotides containing sequences of TT(N)mGCCTC can bind and inhibit archaeal family B DNA polymerases but not Taq DNA polymerase. It is very likely that TT(N)mGCCTC inhibits thermostable DNA polymerases during PCR in a single-stranded form. We showed that guanine-rich sequences such as GGGGG and GGGGHGG can inhibit PCR using family B DNAPs but not Taq DNAP16. We found that oligonucleotides containing TT(N)mGCCTC can bind to archaeal family B DNA polymerases and inhibit their polymerase activity. Unlike the previous identified aptamers[17,18,19], which only inhibit DNAPs at lower temperature, the inhibitory effect of TT(N)mGCCTC to archaeal family B DNAPs persisted during PCR, suggesting TT(N)mGCCTC inhibit archaeal family B DNAPs independent of its secondary structure
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