Reverse Transcriptase
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(Redirected from Reverse transcriptase)
Characteristics of RT
- RNA dependent, DNA polymerase
- Requires a suitable primer
- Sequence specific
- Random hexamers
- Poly-U (in Eukaryotes)
- Two functional domains:
- DNA polymerase
- RNase H
- Naturally occuring in RNA viruses:
- AMV = Avian Myeloblastosis Virus
- Mo-MLV = Moloney strain Murine Leukemia Virus
- HIV
- Most commercial RTs are either AMV or Mo-MLV derivatives
AMV vs. Mo-MLV
Mo-MLV AMV Single polypeptide chain 2 polypeptide chains 2 independently functioning, non-overlapping domains Amino terminal (450 aa) = DNA pol
Carboxyl terminal (220 aa) = RNase H
RNase H and DNA polmerase activity associated with both chains 37-42°C (up to 50°C) 37-45°C (up to 55-60°C) pH .6 pH 8.3 Cloned in 1985 Cloned in 1988 RNase H- available in 1987 RNase H- available in 1998
Problems associated with RT enzymes
- High Error Rate
- Sluggish and inefficient (at best 50% efficient)
- RNase H degradation
- Competition for degradation vs. extension during initial primer binding
- Premature termination
- Prone to Pausing
- Sites rich in Secondary Structure
- Homopolymer runs (particularily A)
- Sensitive to [dNTP]
- RNase H degradation
Alternatives
Thermus thermophilus DNA polymerase (Tth pol)
- Possesses RT activity in the presence of MnCl
- Synthesizes DNA from both RNA and DNA templates
- Mn+ lowers the fidelity of DNA synthesis during PCR
- Thermostable
- Requires sequence specific primer to insure stable binding at elevated temperatures.
- Only capable of producing short products (~ 1-2 kb)