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Detection Limit of High-Fidelity DNA Polymerases on Human gDNA

High-Fidelity DNA Polymerase Comparison
Published on Jan 30, 2017 by Simon Roy, Ph.D
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Detection Limit of High-Fidelity DNA Polymerases using Human gDNA for Template

We previously determined the detection limit of different Taq DNA Polymerases using human genomic DNA as a template for PCR. For this instance, we aimed at determining the Detection Limit of High-Fidelity DNA Polymerases in similar conditions as described previously.

In the current assay, we performed multiplex PCR amplification or a human β-globin fragment (536 bp) and the full-lenght coding region of the Melanocortin-2 Receptor (MC2R; 891 bp) from purified human genomic DNA.

To determine the Detection Limit of High-Fidelity DNA Polymerases, the following masses of purified human genomic DNA were used: 25 ng, 2.5 ng, 250 pg or 25 pg.

KP*, KKP*, FastPfu and FastPfu FLY High-Fidelity DNA Polymerases and competitor DNA Polymerases: Phusion® HotStart II and Q5® High-Fidelity DNA Polymerases were compared.

Details

Date: January 29th, 2016
Type of experiment: Multiplex PCR from human gDNA
DNA Polymerase: KP*, KKP*, TransStart FastPfu and FastPfu FLY High-Fidelity DNA Polymerases

Competitor DNA Polymerases: Phusion® HotStart II and Q5® High-Fidelity DNA Polymerases

Phusion® is a registered trademark of ThermoFisher Scientific. Q5® is a registered trademark of New England Biolabs, Inc.

*KP and KKP are currently prototype versions of 2 new High-Fidelity DNA Polymerases that are under development.

Multiplex PCR setup for Detection Limit of High-Fidelity DNA Polymerases

  • H2O : fill to 15 ul
  • 5x buffer : 3 ul
  • dNTPs (2.5 mM): 1.2 ul (0.2 mM final)
  • β-globin Forward primer (10 uM): 0.3 ul (0.2 uM final)
  • β-globin Reverse primer (10 uM) : 0.3 ul (0.2 uM final)
  • MC2R Forward primer (10 uM): 0.3 ul (0.2 uM final)
  • MC2R Reverse primer (10 uM) : 0.3 ul (0.2 uM final)
  • gDNA : 1 ul (sequencially pre-diluted to 25000, 2500, 250 and 25 pg per ul)
  • DNA Polymerase (2.5 u/ul) : 0.3 ul

Multiplex PCR setup for Detection Limit of High-Fidelity DNA Polymerases

  • H2O : fill to 15 ul
  • 5x buffer : 3 ul
  • dNTPs (2.5 mM): 1.2 ul (0.2 mM final)
  • β-globin Forward primer (10 uM): 0.75 ul (0.5 uM final)
  • β-globin Reverse primer (10 uM) : 0.75 ul (0.5 uM final)
  • MC2R Forward primer (10 uM): 0.75 ul (0.5 uM final)
  • MC2R Reverse primer (10 uM) : 0.75 ul (0.5 uM final)
  • gDNA : 1 ul (sequencially pre-diluted to 25000, 2500, 250 and 25 pg per ul)
  • DNA Polymerase (2 u/ul) : 0.15 ul for Phusion and Q5; 0.3 ul for KP

Multiplex PCR cycling for establishing the Detection Limit of High-Fidelity DNA Polymerases

Denaturation: 60s at 95 °C

35 x

  • Denaturation: 15s at 95 °C
  • Annealing: 20s at 57 °C (KP, KKP, FastPfu, FLY) or 60 °C (Phusion, Q5)
  • Extension: 25s at 72 °C

Final extension: 120s at 72 °C

Detection Limit of High-Fidelity DNA Polymerases 25ng to 25pg Phusion vs Q5

Phusion and Q5 Detection Limit on gDNA

First, the Detection Limit of High-Fidelity DNA Polymerase multiplex PCR was performed using competitor DNA Polymerases, namely Phusion® HotStart II and Q5® High-Fidelity DNA Polymerases. The annealing temperature used was 60 °C.

First, Phusion® was more specific than Q5®, although offering less DNA yield. However, Phusion® HotStart II and Q5® were able to amplify bands corresponding to human β-globin (536 bp) and MC2R (891 bp) using 25 ng, 2.5 ng and 250 pg of human genomic DNA. These DNA masses correspond to 7.5 x 103, 7.5 x 102 and 7.5 x 101 copies of target template respectively. Abruptly, Phusion® stopped ‘working’ with 25 pg gDNA, whereas Q5® detected β-globin. However, MC2R amplification using Q5 was very modest and unefficient using only 25 pg gDNA (7.5 copies).

Compare DNA Polymerases

FastPfu and FastPfu FLY Detection Limit on gDNA

Secondly, Detection Limit of High-Fidelity DNA Polymerase multiplex PCR was performed using TransStart FastPfu and FastPfu FLY High-Fidelity DNA Polymerases. The annealing temperature used to obtain this result was 57 °C.

First, FastPfu gave similar DNA yield for MC2R amplification s compared FastPfu FLY using 57 °C as the annealing temperature. Nevertheless, FastPfu could detect human β-globin (536 bp) using all the gDNA input range.

Remarkably, FastPfu FLY was very specific in addition to successfully amplifying both β-globin (536 bp) and MC2R (891 bp). Each band (8 ul load) represently approximately 50 ng as compared to the 100bp DNA ladder. Simply superb performance (again)!

FLY, FLY, FLY!
Detection Limit of High-Fidelity DNA Polymerases 25ng to 25pg FastPfu vs FLY
Resume Detection Limit of High-Fidelity DNA Polymerases 25ng vs 25pg

Summary of the Detection Limit of High-Fidelity DNA Polymerases

In one image, we recapitulated the results of the performance comparison and the Detection Limit of High-Fidelity DNA Polymerases on human gDNA. All amplicons obtained using either 25 ng or 25 pg with all tested High-Fidelity DNA Polymerase could then be compared side-by-side.

This comparison better illustrates the differences between each DNA Polymerase.

Ranking of High-Fidelity DNA Polymerases:

  • Detection limit: FLY > Q5 = KKP > FastPfu > KP > Phusion
  • Yield: FLY > Q5 ≥ KKP > KP ≥ FastPfu ≥ Phusion
FLY = Higher Performance

Setting a Higher Level of DNA Polymerase Performance

Clearly, our results demonstrate that TransBionovo High-Fidelity DNA Polymerases set a Higher Level of DNA Polymerase Performance when compared to the popular products such as Phusion® and Q5® DNA Polymerases.

The WINNER of this ” Detection Limit of High-Fidelity DNA Polymerases ” comparison is: TransStart FastPfu FLY (Ultra-HiFi )DNA Polymerase.

In addition, KP and KKP DNA Polymerases, currently in development, have shown promising performance, albeit not being able to outperform the great FastPfu FLY!

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