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Mutational analysis using denaturing gradient gel electrophoresis and PCR

N F Cariello¹, T R Skopek

¹University of North Carolina, Pathology Department, Chapel Hill 27599.

Mutation Research

|July 1, 1993

Summary

This study combines Polymerase Chain Reaction (PCR) with Denaturing Gradient Gel Electrophoresis (DGGE) for precise DNA mutation analysis. The PCR-DGGE method effectively identifies sequence variations and purifies mutant DNA, proving valuable in genetic research.

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Area of Science:

Molecular Biology
Genetics
Biochemistry

Background:

Denaturing Gradient Gel Electrophoresis (DGGE) separates DNA fragments based on sequence differences.
Polymerase Chain Reaction (PCR) enables rapid amplification of specific DNA regions.

Purpose of the Study:

To demonstrate the utility of combining PCR and DGGE for comprehensive mutation analysis.
To showcase the application of this combined technique in various genetic contexts.

Main Methods:

Utilizing DGGE under specific conditions to resolve base-pair substitutions from wild-type DNA.
Employing PCR for rapid amplification of target genomic regions for subsequent analysis.
Applying the combined PCR-DGGE technique to analyze mutations in mouse p53 cDNA and HPRT exon 3.

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Assessing the fidelity of Pyrococcus furiosus (Pfu) DNA polymerase.

Purifying mutant DNA from wild-type DNA contamination in mouse T-cell clones.

Main Results:

Successful identification of sequence variations using the PCR-DGGE approach.
Demonstrated ability to analyze mutations in mouse p53 cDNA and HPRT exon 3.
Evaluation of DNA polymerase fidelity.
Effective purification of mutant DNA from mixed samples.

Conclusions:

The combination of PCR and DGGE is a powerful tool for sensitive DNA mutation detection and analysis.
This method offers broad applicability in genetic research, including mutation screening, fidelity assessment, and DNA purification.