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Primer Extension Reactions for the PCR- based α- complementation Assay.
Vasudevan Achuthan1, Jeffrey J DeStefano1
1Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, USA.
Bio-Protocol
|August 9, 2016
Summary
The PCR-based α-complementation assay accurately measures polymerase fidelity, including RNA-dependent RNA polymerases and Reverse Transcriptases. This method is crucial for understanding enzyme accuracy in processes like reverse transcription.
Area of Science:
- Molecular Biology
- Enzymology
Background:
- Polymerase fidelity is critical for genetic stability and accurate replication.
- Existing methods for measuring polymerase fidelity can be limited in sensitivity and scope.
Purpose of the Study:
- To detail the methodology for preparing inserts for the PCR-based α-complementation assay.
- To highlight the utility of this assay for measuring the fidelity of various polymerases, particularly Reverse Transcriptases.
Main Methods:
- Utilizing a PCR-based α-complementation assay.
- Preparing DNA inserts via primer extension reactions.
- Measuring low yields of RNA synthesis or reverse transcription products.
Main Results:
- The assay effectively measures polymerase fidelity, including RNA-dependent RNA polymerases and Reverse Transcriptases.
- Demonstrated the accuracy of HIV Reverse Transcriptase at physiological magnesium concentrations.
Conclusions:
- The PCR-based α-complementation assay is a sensitive and versatile tool for assessing polymerase fidelity.
- The assay provides insights into the accuracy of enzymes involved in crucial biological processes like reverse transcription.
