Reverse Transcriptase

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Contents

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)
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