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Recombinant HIV Reverse Transcriptase
- 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, 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 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.
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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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