Simply ‘FLY’ your long PCRs – Polymerase Extension Rate
Transgen Biotech manufactures three High-Fidelity DNA Polymerases, TransStart® KD Plus, FastPfu and FastPfu FLY. TransStart® FastPfu FLY, a ultra-high fidelity (108x higher fidelity than EasyTaq) and ultra-high processivity DNA polymerase is claimed to provide efficient PCR amplification and a Polymerase Extension Rate of 6 kb/min (10 s/kb). TransStart® FastPfu, a high-fidelity (54x higher fidelity than EasyTaq) also is very processive, allowing an efficient Polymerase Extension Rate up to 4 kb/min (15 s/kb).
As their exclusive Canadian distributor and as scientists ourselves, Civic Bioscience developped a test to assess Polymerase Extension Rate. Our unique Polymerase Extension Rate assay is based on the pcDNA5/FRT plasmid containing different constructs between the TATA-box and BGH polyA signal. The primers used were originally designed by Roy et al. 2007 to amplify XhoI-SphI-TATA box-cytomegalovirus-GOI-polyA-SphI-SpeI in order to construct bicistronic vectors containing Myc-tagged MC2R (melanocortin-2 receptor) and MRAPα-Flag (melanocortin receptor accessory protein isoform α). We have re-edited the original primers used in this study by removing the forward primer’s Xho I and Sph I restriction sites and 2 out of 6 bases of the reverse primer’s Sph I site. Thereby, our primers have a 100% homology match to the pcDNA5/FRT backbone template. Forward (pc5cis forward) and reverse (pc5cis reverse) primers are as follows: 5′-CAATTGCATGAAGAATCTGC-3′ and 5–ATGCCTGCTATTGTCTTCC-3′.
In order to test wether TransStart® FastPfu FLY could amplify DNA at a rate of 6 kb/min, we eliminated the variability of PCR primer GC percentage, secondary structure and annealing temperatures using the pc5cis primer pair but also by keeping the exact same vector backbone. We chose pcDNA5/FRT as we possessed the empty vector along with a multitude of pcDNA5/FRT constructs containing different inserts between the vector’s TATA box and BGH polyA signal. In addition, we tested TransStart® FastPfu in the exact same conditons as FastPfu FLY.
- pcDNA5-FRT : 1067 bp; minimal elongation of 10,7 s at 6 kb/min and 16 s at 4 kb/min.
- pcDNA5/FRT/Flag-MRAPα : 1597 bp; minimal elongation of 16 s at 6 kb/min and 24 s at 4 kb/min.
- pcDNA5/FRT/Myc-MC2R : 1985 bp; minimal elongation of 19,9 s at 6 kb/min and 29,8 s at 4 kb/min.
- pcDNA5/FRT/Halo-Myc-MC2R: 2873 bp; minimal elongation of 28,7 s at 6 kb/min and 43,1 s at 4 kb/min.
The extension time used for PCR cycling was 29 s. At a Polymerase Extension Rate of 6 kb/min, 29 seconds is sufficient for FastPfu FLY, but not FastPfu, to amplify a 2873 bp amplicon from pcDNA5/FRT/Halo-Myc-MC2R.
FLY vs FastPfu Polymerase Extension Rate
- pc5 = pcDNA5-FRT : 1067 bp;
- 5/Flag-MRAPα : 1597 bp;
- 5/Myc-MC2R : 1985 bp;
- 5/Halo-Myc-MC2R: 2873 bp.
25 ul reaction setup: at room temperature and as recommended by the TransStart® FastPfu FLY’s and FastPfu‘s datasheets without any additives and using 10 ng vector as templates. 5 ul of each reaction was loaded in each well.
- Denaturation : 120 s at 95 °C
- 35 cycles:
- 15 s at 95 °C
- 20 s at 51 °C
- 29 s at 72 °C
- Final extension: 45 s at 72 °C
As scientists ourselves, we assesed wether FastPfu FLY DNA Polymerase could perform according to its specification sheet. The expected Polymerase Extension Rate was 6 kb/min. We found that FastPfu FLY remarkedly amplified all targets with high yield. To our surprise, although it offered a fraction less PCR yield, FastPfu was also able to amplify the longest target of 2873 bp. This is better than stated in its own specification sheet (4 kb/min).
Optimal results were obtained with TransStart® FastPfu FLY DNA Polymerase in our Polymerase Extension Rate assay. FastPfu FLY could amplify our targets at at least 6 kb/min.
Next, we will assess wether TransFast® Taq is also up to the 6 kb/min performance test. Preliminary results indicate that it can easily withstand 4 kb/min…
But for the moment.. FLY FastPfu FLY !