Many ribonucleic acid (RNA) molecules contain secondary structure that results from hybridization between complementary regions within the RNA molecule. A variety of secondary structures can be formed, including hairpins and cruciforms. RNA molecules containing secondary structure are often difficult to reverse transcribe because polymerases cannot readily process through the secondary structure.
Because of the difficulty of reverse transcribing RNA molecules with secondary structure, many techniques dependent upon reverse transcription yield anomalous results. For example, RNA molecules with secondary structure may be poorly represented in cDNA libraries. Populations of RNA with secondary structure may also yield cDNA libraries with a short insert size. Furthermore, RNA molecules containing secondary structure may be difficult to detect in assays such as reverse transcription-polymerase chain reaction (RT-PCR).
Traditionally, reverse transcription has been performed with reverse transcriptases encoded by retroviruses (e.g., avian myoblastosis virus (AMV) reverse transcriptase and Moloney murine leukemia virus (MMLV) reverse transcriptase). Several mesophillic DNA polymerases (e.g., E. coli DNA polymerase I) have also been shown to possess reverse transcriptase activity. However, these enzymes are generally used at temperatures of between about 37° C. to 42° C., a temperature range where secondary structure can be a significant problem.
Several thermophilic DNA polymerases (e.g., Thermus aquaticus DNA polymerase and Thermus thermophilus DNA polymerase) also have reverse transcriptase activity. These enzymes are useful for reverse transcription, because at the high temperatures where such enzymes are stable, secondary structure in RNA molecules is reduced. Furthermore, such enzymes can be used to directly synthesize second strand DNA and potentially even to directly amplify an RNA target. However, the utility of these thermostable enzymes is limited because they require manganese as a co-factor for reverse transcriptase activity (e.g., U.S. Pat. No. 5,322,770) resulting in deleterious effects. In some cases, the fidelity of the polymerase is reduced as compared to the fidelity of the enzyme in the presence of other cofactors, such as magnesium ions. Therefore, it is not desirable to amplify the template in the same reaction mixture in which reverse transcription reaction is conducted. This necessitates extra time consuming steps when performing RT-PCR. In other cases, the presence of manganese ions may also cause degradation of the RNA template.
Accordingly, what is needed in the art are alternative thermostable polymerases that have reverse transcriptase activity. Preferably, such thermostable polymerases should have reverse transcriptase activity in the presence of magnesium so that high-fidelity cDNAs may be obtained and so that both reverse transcription and amplification in RT-PCR reactions may conducted in the same reaction mixture.