Avian myeloblastosis virus reverse transcriptase is easier to use than the Klenow fragment of DNA polymerase I for labeling the 3′-end of a DNA fragment

Abstract

The 3′→5′ exonuclease activity of the Klenow fragment operates in 3′-end labeling of DNA fragments. In the presence of excess deoxyribonucleoside 5′-triphosphates (dNTPs), the 5′→3′ polymerase activity is dominant over the exonuclease activity. However, in the presence of a small amount of dNTPs, the exonuclease activity removed deoxyribonucleoside 5′-monophosphate (dNMP) incorporated in the 3′-end of a DNA strand by the polymerase activity. We found that the radioactivity of incorporated dNMP decreased remarkably in the course of 3′-end labeling by the Klenow fragment. On the other hand avian myeloblastosis virus (AMV) reverse transcriptase also possesses the polymerase activity. The decline of the incorporated radioactivity was not observed, indicating that the enzyme has neither exo- nor endonuclease activities. Further-more, the level of the incorporated radioactivity was the same as that obtained by the Klenow fragment. We conclude that AMV reverse transcriptase is easier to use than the Klenow fragment for labeling the 3′-end of a DNA fragment.