Long Range PCR

Long range PCR typically refers to the amplification of DNA fragments in excess of 5 kb, using a blend of thermostable DNA polymerases that allows for the amplification of longer fragments than those that can be achieved with a single enzyme. Enzyme blends consist primarily of an A-family DNA polymerase (e.g. Taq), combined with a small amount of a B-family or proofreading DNA polymerase (e.g. Pfu). Both enzymes possess 5’ – 3’ DNA-dependent DNA polymerase activity, but only the B-family polymerase possesses 3’ – 5’ exonuclease or proofreading activity.

A-family DNA polymerases like Taq are unable to efficiently extend beyond misincorporated bases due to their lack of proofreading activity. Mismatched base pairing therefore leads to truncated products that accumulate during PCR, reduce the effective template copy number and contribute to reaction failure if the target is long and/or the concentration of template DNA is low. Wild-type B-family DNA polymerases are extremely accurate due to their ability to repair mismatches but do not perform well over longer target distances or with low template concentrations because their 3’ – 5’ exonuclease (proofreading) activity destroys primers and affects sensitivity. By combining the robustness of A-family polymerases with misincorporation repair through the 3’ - ‘5’ exonuclease activity of the minor (B-family polymerase) component, long-range blends are capable of long and sensitive PCR. The error rates of enzyme blends are typically two- to six-fold lower than that of Taq, but are significantly higher than that of pure B-family DNA polymerases.

KAPA Long Range DNA Polymerase is a blend of KAPA Taq DNA polymerase and the novel, engineered KAPA HiFi B-family DNA polymerase. In the HotStart formulation, KAPA Taq is combined with a proprietary antibody that blocks Taq activity until the initial denaturation step. This prevents the formation of spurious reaction products during reaction setup, and increases overall reaction efficiency.

KAPA Long Range and KAPA Long Range HotStart PCR Kits offer:

  • Efficient amplification of long DNA fragments (up to 25 kb).
    Improved sensitivity in the amplification of both short and long amplicons.
    Two- to six-fold higher in fidelity than wild-type Taq.
    The robustness of A-family polymerases, i.e. typically less optimization of reaction parameters than required for B-family polymerases.
    dA-tailed reaction products that can be cloned into TA-cloning vectors.