Patterns of PCR Amplification Artifacts of the Fungal Barcode Marker in a Hybrid Mushroom
Related Experiment Video
Updated: Jan 2, 2026
Isolation, Characterization, and Total DNA Extraction to Identify Endophytic Fungi in Mycoheterotrophic Plants
Published on: May 5, 2023
Jun-Liang Zhou1,2, Jianping Xu3,4, An-Guo Jiao1
1Institute of Plant and Environment Protection, Beijing Academy of Agriculture and Forestry Sciences, Beijing Engineering Research Center for Edible Mushroom, Beijing, China.
Abstract:
The polymerase chain reaction (PCR) is widely used in modern biology and medicine. However, PCR artifacts can complicate the interpretation of PCR-based results. The internal transcribed spacer (ITS) region of the ribosomal RNA gene cluster is the consensus fungal barcode marker and suspected PCR artifacts have been reported in many studies, especially for the analyses of environmental fungal samples. At present, the patterns of PCR artifacts in the whole fungal ITS region (ITS1+5.8S+ITS2) are not known. In this study, we analyzed the error rates of PCR at three template complexity levels using the divergent copies of ITS from the mushroom Agaricus subrufescens. Our results showed that PCR using the Phusion® High-Fidelity DNA Polymerase has a per nucleotide error rate of about 4 × 10-6 per replication. Among the detected mutations, transitions were much more frequent than transversions, insertions, and deletions. When divergent alleles were mixed as templates in the same reaction, a significant proportion (∼30%) of recombinant molecules were detected. The in vitro mixed-template results were comparable to those obtained from using the genomic DNA of the original mushroom specimen as template. Our results indicate that caution should be in place when interpreting ITS sequences from individual fungal specimens, especially those containing divergent ITS copies. Similar results could also happen to PCR-based analyses of other multicopy DNA fragments as well as single-copy DNA sequences with divergent alleles in diploid organisms.
Insights
Polymerase chain reaction (PCR) can introduce artifacts, especially in fungal ITS sequencing. This study quantifies PCR errors and recombination, revealing significant artifact rates that necessitate careful interpretation of fungal DNA sequences.
Area of Science:
- Molecular Biology
- Mycology
- Genetics
Background:
- Polymerase chain reaction (PCR) is a cornerstone of modern biology and medicine.
- PCR artifacts can compromise the accuracy of PCR-based results, particularly in complex environmental fungal samples.
- The internal transcribed spacer (ITS) region is the standard fungal DNA barcode, but its susceptibility to PCR artifacts is not fully understood.
Purpose of the Study:
- To investigate the error rates and artifact patterns of PCR in the fungal ITS region.
- To analyze the impact of template complexity on PCR error rates.
- To assess the frequency of recombinant molecules generated during PCR amplification of divergent ITS alleles.
Main Methods:
- Utilized Phusion® High-Fidelity DNA Polymerase for PCR amplification.
- Analyzed PCR error rates using divergent ITS copies from *Agaricus subrufescens* at three template complexity levels.
- Quantified mutation types (transitions, transversions, insertions, deletions) and assessed recombinant molecule formation.
Main Results:
- The per nucleotide error rate of the polymerase was determined to be approximately 4 × 10⁻⁶ per replication.
- Transitions were the predominant mutation type, occurring more frequently than transversions, insertions, or deletions.
- A substantial proportion (approximately 30%) of recombinant molecules were detected when divergent ITS alleles were mixed as templates.
Conclusions:
- Caution is advised when interpreting fungal ITS sequences, especially from specimens with divergent ITS copies.
- PCR-generated artifacts, including recombination, can significantly impact sequence data.
- These findings have implications for PCR-based analyses of other multicopy DNA fragments and diploid organisms with divergent alleles.
