Reverse transcriptase adds nontemplated nucleotides to cDNAs during 5 '-RACE and primer extension

In determining the terminal sequences of the genomic dsRNAs of rotavirus by 5'-rapid amplification of cDNA ends (5'-RACE), it was found that most of t he viral cDNAs contained extra nucleotides at their 5' termini that had not been reported before on any rotavirus sequence. Although the extra nucleot ides could be dA, dC, dG, or dT residues, the extra nucleotides an the cDNA s usually consisted of a single dr residue. Experiments performed with DNA/ RNA duplexes indicated that reverse transcriptase has an associated termina l nucleotidyl transferase (TdT)-like activity, which can add nontemplated n ucleotides to the 3' ends of DNA, and that reverse transcriptase was respon sible for the presence of the extra nucleotides detected on the 5'-RACE cDN As. The TdT-like activity of reverse transcription was specific for double- stranded substrates (i.e., DNA/DNA or DNA/RNA duplexes) and was active over a wide range of temperatures and enzyme concentrations. Both commercially available Moloney murine leukemia virus and avian myeloblastosis virus reve rse transcriptases contained the TdT-like activity. This work implies that 5'-RACE and primer extension assays must be used carefully in determining t he terminal sequences of nucleic acids because, under standard reaction con ditions, reverse transcriptase can add nontemplated nucleotides ra the 3' e nds of cDNAs following template-directed synthesis.