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Temperature-dependent template switching during in vitro cDNA synthesis by the AMV-reverse transcriptase.

M Ouhammouch1, E N Brody

  • 1CNRS Centre de Génétique Moléculaire, Laboratoire Propre Associé à l'Université Pierre et Marie Curie, Gif-sur-Yvette, France.

Nucleic Acids Research
|October 25, 1992
PubMed
Summary

Avian myeloblastosis virus reverse transcriptase can switch templates during cDNA synthesis. This template switching is homology-dependent and temperature-dependent, efficiently creating chimeric DNA at high temperatures.

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Area of Science:

  • Molecular Biology
  • Virology
  • Biochemistry

Background:

  • Reverse transcriptase template switching is a known phenomenon in retroviral replication and recombination.
  • This process has been previously observed in vitro with Moloney murine leukemia virus (M-MuLV) reverse transcriptase.

Purpose of the Study:

  • To investigate the in vitro template switching capabilities of avian myeloblastosis virus (AMV) reverse transcriptase.
  • To determine the conditions influencing AMV reverse transcriptase template switching during cDNA synthesis.

Main Methods:

  • In vitro cDNA synthesis using AMV reverse transcriptase.
  • Incubation of reactions at varying temperatures (37°C to 50°C) and time points (30 min to 90 min).
  • Analysis of generated cDNA products for chimeric molecules.

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Main Results:

  • AMV reverse transcriptase demonstrated template switching in a homology-dependent and temperature-dependent manner.
  • Chimeric cDNA molecules were generated efficiently within 30 minutes at high temperatures (42°C–50°C).
  • Detection of chimeric products required longer incubation times at lower temperatures (90 min at 37°C).

Conclusions:

  • Avian myeloblastosis virus reverse transcriptase exhibits template switching during in vitro cDNA synthesis.
  • High temperatures and homology between templates enhance the efficiency and speed of chimeric cDNA formation.
  • Understanding this mechanism is crucial for retroviral replication and recombination studies.