Choice Of RT-PCR Enzymes

Obviously, in RT-PCR a major factor to consider is the choice of reverse transcriptase used to synthesize cDNA. Since each of the available enzymes has different enzymatic properties, one may be more suitable for a specific experiment than the others. Among the enzyme properties to consider are:

Temperature optima

Higher incubation temperatures can help eliminate problems of template secondary structure. In addition, high temperature improves the specificity of reverse transcription by decreasing false priming. Thus, thermoactive reverse transcriptases that can be incubated at high temperatures (50°–70°C) are more likely to produce accurate copies of mRNA, especially if the template has a high GC content.

Note: At these high temperatures, use only specific primers; do not use oligo(dT) or random hexamer primers.

RNase H activity

In contrast to Tth DNA Polymerase, AMV and M-MuLV Reverse Transcriptases have RNase H activity. This activity will specifically degrade RNA in an RNA:cDNA hybrid. This can be detrimental if degradation of template RNA competes with DNA synthesis during production of first-strand cDNA. M-MuLV Reverse Transcriptase has lower RNase H activity than AMV Reverse Transcriptase. Mutated enzymes without RNase H activity are also available, e.g. Expand Reverse Transcriptase (Cat. No. 1785826) or the C. therm. Polymerase for Reverse Transcription in Two-Step PCR (Cat. No. 2016311) from Roche Applied Science.

Note: Expand Reverse Transcriptase is not for sale in the United States.

Divalent ion requirement

Most reverse transcriptases require a divalent ion for activity. Enzymes that use Mg²⁺ are likely to produce more accurate cDNA copies than those that use Mn²⁺, since Mn²⁺ adverselyaffects the fidelity of DNA synthesis.

Specificity and sensitivity

Reverse transcriptases have differing ability to copy small amounts of template (sensitivity). They also differ in their ability to transcribe RNA secondary structures accurately (specificity).

Reverse transcriptase – DNA polymerase combinations

Using different reverse transcriptase – DNA polymerase combinations can allow you to optimally amplify certain length targets.

The properties of reverse transcriptases and their suggested applications are given in the tables listed below.

		Properties of reverse transcriptases
		Suggested applications for Reverse Transcriptases
		Reverse transcriptases suitable for one-step RT PCR
		Reverse transcriptases suitable for two-step RT PCR