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FastPfu FLY Ultra-HiFi DNA Polymerase Extension Rate

The 6 kb/min Polymerase Extension Rate
More about FastPfu FLY

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.

Using different constructs (Roy et al. 2007, Roy et al. 2011), the lenght of dsDNA and minimal Polymerase Extension Rate expected are as follows:

  • 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.

 

FastPfu FLY and FastPfu 29s Polymerase Extension Rate

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.

PCR cycling:

  1. Denaturation : 120 s at 95 °C
  2. 35 cycles:
    • 15 s at 95 °C
    • 20 s at 51 °C
    • 29 s at 72 °C
  3. 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 !