• More thermostable than AMV RT and M-MLV RT
  • Excellent target to evaluate antiviral agents
  • Highly sensitive enzyme for RT-PCR applications
  • Ideal for reverse transcribing transcripts with high G/C content

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, Mg 2+, 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 37°C and 42°C. However, the enzyme retains a significantly higher fraction of its activity at 50°C 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 supplied with the enzyme.

Figure 1. HIV RT Outperforms MMLV and AMV RT Enzymes in cDNA Synthesis at 50ºC. A synthetic RNA transcript for Xef-1 (1 µg) was reverse transcribed with oligo(dT) primers in a 20 µl reaction using 5 µg/µl M-MLV RT (Ambion), 0.5 U/µl AMV RT (Seikagaku), or 0.5 µg/µl 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/µl ribonuclease inhibitor. The percentage cDNA conversion was determined by measuring the amount of cDNA made via scintillation counting.

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References

  1. Sorbo BH (1953) Acta Chem Scand 7: 1129.

  2. Kudlicki W, Odom OW, Kramer G, Hardesty B (1994) Activation and release of enzymatic ally inactive, full-length Rhodanese that is bound to ribosomes as peptide-tRNA. J Biol Chem 269(24): 16549-165