Use of avian myeloblastosis virus reverse transcriptase at high temperature for sequence analysis of highly structured RNA

Avian myeloblastosis virus reverse transcriptase (AMV RT) is routinely used in the sequence analysis of RNA and DNA templates. We review the various methods for dealing with secondary structures that would otherwise result in premature termination or sequence compression. Based on our experience in sequencing the 11-kb single-stranded RNA genome of lymphocytic choriomeningitis virus, we have found that raising the reaction temperature above 47°C is the simplest way to overcome template secondary structure, and the use of 98% formamide gels is the simplest way to overcome product secondary structure.