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Related Concept Videos

Mismatch Repair01:20

Mismatch Repair

7.0K
Organisms are capable of detecting and fixing nucleotide mismatches that occur during DNA replication. This sophisticated process requires identifying the new strand and replacing the erroneous bases with correct nucleotides. Mismatch repair is coordinated by many proteins in both prokaryotes and eukaryotes.
The Mutator Protein Family Plays a Key Role in DNA Mismatch Repair
The human genome has more than 3 billion base pairs of DNA per cell. Prior to cell division, that vast amount of genetic...
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Correction for Biswas et al., "A novel aptamer-based dNTP assay reveals that intact HIV virions are highly stable and do not contain enough dNTPs to support DNA synthesis".

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Related Experiment Video

Updated: Mar 22, 2026

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis
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Mismatched Primer Extension Assays.

Vasudevan Achuthan1, Jeffrey J DeStefano1

  • 1Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, USA.

Bio-Protocol
|April 16, 2016
PubMed
Summary

This study details a mismatched primer extension assay for measuring human immunodeficiency virus reverse transcriptase (HIV RT) fidelity. The assay quantifies polymerase extension efficiency at specific Mg2+ concentrations, aiding in enzyme fidelity studies.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Steady-state kinetic assays are established methods for estimating polymerase fidelity.
  • Mismatched primer extension assays allow analysis of primer extension with 3' mismatches.
  • Previous work demonstrated increased HIV RT fidelity at low Mg2+ concentrations (~0.25 mM).

Purpose of the Study:

  • To provide a detailed protocol for the mismatched primer extension assay.
  • To measure the standard extension efficiency of HIV RT at 2 mM Mg2+.
  • To enable estimation of relative polymerase fidelity using this assay.

Main Methods:

  • Detailed description of the mismatched primer extension assay protocol.
  • Utilizing human immunodeficiency virus reverse transcriptase (HIV RT).

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  • Assay performed at a standard Mg2+ concentration of 2 mM.

    • Main Results:

    • The described method allows for the measurement of standard extension efficiency.
    • This efficiency is used to estimate the relative fidelity of HIV RT.
    • The protocol is based on established methods (Mendelman et al., 1990).

    Conclusions:

    • The mismatched primer extension assay is a valuable tool for assessing polymerase fidelity.
    • The detailed protocol facilitates reproducible measurements of HIV RT extension efficiency.
    • This method contributes to understanding enzyme fidelity under specific conditions.