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Choosing the Correct Enzyme for PCR

correct enzyme

A wide variety of enzymes are available for PCR and RT-PCR and the optimal choice depends on a range of factors specific to your experiment. Some of these factors will now be explored to help you to make the most suitable and cost effective choices when ordering.

PCR Type and Other Factors to Consider

First, consider the type of PCR you are doing. These include: standard/conventional, hot start, long, high fidelity, high specificity, multiplex, and GC-rich PCR.  Choose the enzyme that is best for each application (manufacturer’s inserts can help you choose).  Then look at the following specifics of your PCR.

Amplicon Size

Look at the length of your PCR product. Use standard polymerases for shorter sequences as they are  less expensive. Choose a more expensive polymerase specific for long PCR when needed.

Activation Time

Activation time refers to the length of time that elapses before the enzyme is activated at 95’C. To avoid non-specific amplification of DNA, use ‘hot-start’ enzymes. They are inactive until the temperature is raised and thus no amplification occurs at room temperature or lower during a PCR cycle. While some enzymes are permanently activated after 15 seconds of heat, others require up to 10 minutes.

Degree of Specificity Required

Are you expecting trouble with amplifying the right product?  Choose an enzyme that is highly specific if you require a highly pure sample end product. This is particularly important when the target sequence is in low abundance and if primers are not optimally specific.

Fidelity

Some polymerases replicate the target sequence more faithfully than others. If you need high fidelity, for example in SNP detection, then choose a polymerase with  3’-5’ exonuclease activity.

Degree of Sensitivity Required

Contamination is a huge problem in PCR as very sensitive polymerases amplify minute DNA quantities. Less sensitive polymerases do not detect minute quantities of DNA, so don’t use them when DNA starting quantities are low.

Standard Taq or Hot Start Taq?

For many routine applications, use a standard Taq DNA polymerase. However, Taq has activity below its optimal temperature of 72’C. Therefore, prior to the first heating step, primer-dimers can form (primers binding to one another) or the primers can bind non-specifically to the DNA template. In both scenarios, elongation can happen before the first DNA melting (or denaturation) step has occurred. Both of these problems have a huge effect on product yield, reducing the amount of target sequences synthesized. To avoid this problem, use “hot start” (or FastStart) enzymes (see above).

Be aware:  neither standard Taq nor hot start Taq is capable of proofreading newly synthesized oligonucleotides. This leads to errors and can also cause the enzyme to stall creating shortened oligonucleotide products. When cloning or sequencing, use enzymes with 3’-5’ exonuclease activity. When you need high yield and high fidelity, consider a blended polymerase mix.   These blends are a mixture of enzymes usually containing Taq and a polymerase with proofreading activity.  They are especially helpful for amplification of GC-rich sequences.

Before placing an order, consult the website of the company for detailed information about a polymerase.

Have you any further pointers for enzyme choice?

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