Reverse Transcriptase

Characteristics of RT

 * 1) RNA dependent, DNA polymerase
 * 2) Requires a suitable primer
 * 3) *Sequence specific
 * 4) *Random hexamers
 * 5) *Poly-U (in Eukaryotes)
 * 6) Two functional domains:
 * 7) *DNA polymerase
 * 8) *RNase H
 * 9) Naturally occuring in RNA viruses:
 * 10) *AMV = Avian Myeloblastosis Virus
 * 11) *Mo-MLV = Moloney strain Murine Leukemia Virus
 * 12) *HIV
 * 13) *Most commercial RTs are either AMV or Mo-MLV derivatives

AMV vs. Mo-MLV

 * {| border="1" cellpadding="2" cellspacing="0"

! Mo-MLV || AMV Amino terminal (450 aa) = DNA pol
 * Single polypeptide chain || 2 polypeptide chains
 * 2 independently functioning, non-overlapping domains
 * 2 independently functioning, non-overlapping domains
 * 2 independently functioning, non-overlapping domains

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
 * }
 * 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)
 * 3) *RNase H degradation
 * 4) **Competition for degradation vs. extension during initial primer binding
 * 5) **Premature termination
 * 6) *Prone to Pausing
 * 7) **Sites rich in Secondary Structure
 * 8) **Homopolymer runs (particularily A)
 * 9) **Sensitive to [dNTP]

Alternatives
Thermus thermophilus DNA polymerase (Tth pol)


 * 1) Possesses RT activity in the presence of MnCl
 * 2) *Synthesizes DNA from both RNA and DNA templates
 * 3) *Mn+ lowers the fidelity of DNA synthesis during PCR
 * 4) Thermostable
 * 5) Requires sequence specific primer to insure stable binding at elevated temperatures.
 * 6) Only capable of producing short products (~ 1-2 kb)