Reverse Transcriptase

Characteristics of RT

1. RNA dependent, DNA polymerase
2. Requires a suitable primer
   • Sequence specific
   • Random hexamers
   • Poly-U (in Eukaryotes)
3. Two functional domains:
   • DNA polymerase
   • RNase H
4. 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

1. High Error Rate
2. 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]

Alternatives

Thermus thermophilus DNA polymerase (Tth pol)

1. 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
2. Thermostable
3. Requires sequence specific primer to insure stable binding at elevated temperatures.
4. Only capable of producing short products (~ 1-2 kb)