Member since: 2018
Organization: Broad Institute
Great success rate with the highest fidelity.
Application Area: High fidelity PCR
"Among a lot of high fidelity PCR enzymes, KAPA HiFi gives the best success rate. I have almost succeeded >95% of the time when amplifying my target. There is no mutation in most cases, so you don't need to be afraid of cloning issues."
KAPA HiFi HotStart DNA Polymerase is a novel B-family DNA polymerase, engineered to have increased affinity for DNA, without the need for accessory proteins or DNA binding domains. The intrinsic high processivity of the enzyme results in significant improvement in yield, speed and sensitivity when compared with wild-type B-family DNA polymerases. In addition, the ability to amplify long targets, as well as GC- and AT-rich targets, is significantly improved. The enzyme is combined with a proprietary antibody that inactivates the enzyme until the first denaturation step. This prevents nonspecific amplification during reaction setup, increases sensitivity, and improves reaction efficiency.
In the ReadyMix PCR Kit, KAPA HiFi HotStart DNA Polymerase is supplied in a convenient 2X ReadyMix format, containing all reaction components except primers and template. The ReadyMix contains KAPA HiFi HotStart DNA Polymerase (0.5 U per 25 µL reaction) in a proprietary reaction buffer containing dNTPs (0.3 mM of each dNTP at 1X), MgCl2 (2.5 mM at 1X) and stabilizers.
KAPA HiFi HotStart ReadyMix is designed for routine, high-fidelity PCR of a wide range of targets and fragment sizes. It offers error rates approximately 100 times lower than wild-type Taq DNA polymerase, and higher success rates and yields than achievable with wild-type B-family (proofreading) DNA polymerases. In addition, KAPA HiFi HotStart requires significantly shorter reaction times than wild-type B-family DNA polymerases. KAPA HiFi HotStart DNA Polymerase has 5’g3’ polymerase and 3’g5’ exonuclease (proofreading) activity, but no 5’g3’ exonuclease activity.
The strong 3’g5’ exonuclease activity results in superior accuracy during DNA amplification, lending to KAPA HiFi HotStart DNA Polymerase the lowest published error rate of all B-family DNA polymerases (1 error per 3.6 x 106 nucleotides incorporated). This fidelity is approximately 100 times higher than that of wild-type Taq DNA polymerase, and up to 10 times higher than that of other B-family DNA polymerases and polymerase blends.
DNA fragments generated with KAPA HiFi HotStart ReadyMix may be used for routine downstream analysis and applications, including restriction enzyme digestion, cloning and sequencing. PCR products generated with KAPA HiFi HotStart ReadyMix are blunt-ended, but may be 3’-dA-tailed for cloning into TA cloning vectors (see Important Parameters: TA cloning).