Related Experiment Videos
Low copy number DNA template can render polymerase chain reaction error prone in a sequence-dependent manner
Mansour Akbari1, Marianne Doré Hansen, Jostein Halgunset
1Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, N-7489 Trondheim, Norway.
The Journal of Molecular Diagnostics : JMD
|February 1, 2005
Summary
Low DNA input in polymerase chain reaction (PCR) can create false mutations, particularly guanine to adenine transitions. This PCR artifact mimics real mutations, highlighting the need for caution in genetic analysis.
Area of Science:
- Molecular Biology
- Genetics
- Pathology
Background:
- Paraffin-embedded tissues are crucial for molecular pathology and genetic studies.
- Formalin-fixed, paraffin-embedded (FFPE) tissues present unique challenges for DNA analysis.
Purpose of the Study:
- To investigate apparent base substitutions found in the human uracil-DNA glycosylase (UNG) gene from FFPE gastric tumors.
- To determine if these base substitutions were genuine mutations or artifacts.
Main Methods:
- DNA isolation from microdissected FFPE gastric tumors.
- Mutation analysis of a specific region of the human UNG gene.
- Polymerase chain reaction (PCR) amplification with varying DNA template inputs.
- PCR artifact analysis using homogenous, diluted plasmid DNA.
Main Results:
- Apparent base substitutions in the UNG gene were identified as PCR artifacts, not genuine mutations.
- Low DNA template input in PCR can lead to sequence-dependent false mutations, primarily guanine to adenine transitions.
- The observed artifact mimicked a previously reported UNG gene mutation found in human glioma.
- Genuine mutations were not detected in the UNG gene across 16 analyzed samples.
Conclusions:
- Caution is essential when interpreting PCR-based somatic mutation data from low DNA template amounts.
- Methods designed to enrich rare mutant molecules may inadvertently amplify PCR-generated artifacts.
- The findings underscore the importance of validating PCR results, especially when using degraded DNA sources like FFPE tissues.