Like M-MLV reverse transcriptase (RT) and AMV RT, Ambion's HIV RT synthesizes cDNA from single-stranded RNA template in the presence of primer, Mg2+, and deoxyribonucleotides. HIV-RT is a recombinant enzyme purified to homogeneity from E. coli. HIV RT is a heterodimeric protein which is comprised of a 66 kDa and a 51 kDa subunit.
HIV RT is More Thermostable than M-MLV RT and AMV RT
HIV RT performs as well as M-MLV RT and AMV RT at 37C and 42C. However, the enzyme retains a significantly higher fraction of its activity at 50C than the other RTs. As shown in Figure 1, HIV RT converts a much larger proportion of a synthetic RNA transcript to cDNA than M-MLV RT and AMV RT even after the reaction has progressed for 1 hour. Thus, when working with RNA transcripts that are refractory to efficient cDNA synthesis or when the transcript being studied has a high GC content, HIV RT is a superior alternative to M-MLV RT and AMV RT.
Ambion's HIV RT is a high quality enzyme that has been tested for contaminating endonuclease and exonuclease activity. The enzyme is available in a convenient 500 U size. A 10X Reaction Buffer is suppli ed with the enzyme.
Figure 1. HIV RT Outperforms MMLV and AMV RT Enzymes in cDNA Synthesis at 50C. A synthetic RNA transcript for Xef-1 (1 g) was reverse transcribed with oligo(dT) primers in a 20 l reaction using 5 g/l M-MLV RT (Ambion), 0.5 U/l AMV RT (Seikagaku), or 0.5 g/l HIV RT (Ambion) for the indicated times. Reactions were performed using the manufacturer-supplied reaction buffer in the presence of *32-P dUTP and 0.5 U/l ribonuclease inhibitor. The percentage cDNA conversion was determined by measuring the amount of cDNA made via scintillation counting.