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Eliminating mitochondrial DNA competition for nuclear DNA primers
S Zullo1, J L Kennedy, J Gelernter
1Laboratory of Biochemical Genetics, National Institute of Mental Health Neuroscience Center at St. Elizabeth, Washington, D.C. 20032.
PCR Methods and Applications
|August 1, 1993
Summary
Nuclear DNA primers can unintentionally amplify mitochondrial DNA due to mismatch priming. A new program, OLIGFIND, helps design primers to avoid this common contamination issue in genetic research.
Area of Science:
- Genetics
- Molecular Biology
- Bioinformatics
Background:
- Nuclear DNA (nucDNA) and mitochondrial DNA (mtDNA) amplification using sequence-tagged site (STS) primers can be confounded by unintended mtDNA amplification.
- Mismatch priming, where primers bind to mtDNA with partial homology at their 3' ends, is a significant challenge in PCR-based genetic studies.
Purpose of the Study:
- To investigate the mechanisms of unintended mitochondrial DNA amplification during nuclear DNA targeted PCR.
- To develop a computational tool to prevent mtDNA contamination in nucDNA amplification experiments.
Main Methods:
- Analysis of primer binding sites on mtDNA using sequence identity at the 3' ends.
- Gel isolation and hybridization of PCR products to detect mtDNA contamination.
- Development and application of the OLIGFIND program to identify unique hexamers absent in human mtDNA.
Main Results:
- Unintended amplification of mtDNA occurred in 2 out of 3 studies using nucDNA STS primers.
- Mass action driven by primer molar surplus facilitates mismatch priming on mtDNA.
- The probability of unintended mtDNA amplification can be as high as 1 in 64 primer pairs.
- OLIGFIND identified 104 hexamers absent in human mtDNA out of 4096 possibilities.
Conclusions:
- Unintended mtDNA amplification is a frequent issue in nucDNA PCR experiments.
- Designing STS primers with 3' hexamers absent in mtDNA, identified by OLIGFIND, can prevent contamination.
- OLIGFIND provides a valuable tool for optimizing primer design and ensuring accurate genetic analysis.