PCR Applications

High Fidelity PCR

Patch Things Up

When do you need it?

High fidelity PCR demands a low rate of nucleotide misincorporation (mismatch). Sequence integrity is particularly important in applications involving gene expression, low-copy-number templates, long templates, cDNA library construction, SNP analysis, and site-directed mutagenesis.

How is it achieved?

The intrinsic properties of polymerases, such as extension rates and 3'-5' exonuclease (proofreading) activity directly influence fidelity. In practice, a mixture of high yield but low fidelity polymerases (i.e. Taq), with high fidelity polymerases (i.e. pfu, Vent) can enhance overall amplification performance. External factors like template sequence, buffer composition, and reaction conditions are also important for high fidelity PCR.

What makes our PCR polymerases the best?

Our high performance enzyme mixes and kits are optimized for a variety of high fidelity PCR applications.

Advantage 2 Polymerase Mix contains a blend of our Titanium Taq DNA Polymerase and a small amount of proofreading enzyme, balancing high yield and fidelity. This enzyme is extremely versatile and can amplify a wide range of DNA templates.
Advantage HF 2 PCR Kit combines Advantage 2 with a proprietary mix of dNTPs and a specially formulated buffer that work together to achieve 30-fold higher fidelity than wild-type Taq DNA Polymerase.
Advantage HD Polymerase is a novel PCR enzyme with a robust 3'–5' exonuclease activity that provides superb accuracy, with a low error rate of 1 error per 20 kb (error rate=# misincorporated bases/# bases synthesized).

How to pick the right enzyme?
Polymerase	Application	Fidelity	Amplicon size (kb) Human/plasmid/cDNA	Max GC content	Hot Start	Cloning compatibility
Advantage 2	cDNA amplification and library construction	3X	<6.0/<18.5/0.4–8.5	60%	Yes	TA
Advantage HF 2	Mutation analysis, high fidelity PCR	30X	<3.5/<2.0/<1.3	60%	Yes	TA
Advantage HD	Cloning and library construction, long PCR	20X	<8.5/<28.0/<4.0	60%	Yes	Blunt
* Relative fidelities are represented as fold improvements over Titanium Taq DNA Polymerase, which has an error rate of 1 mismatch per kb of synthesized DNA.