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Patterns of PCR Amplification Artifacts of the Fungal Barcode Marker in a Hybrid Mushroom. | LitMetric

Patterns of PCR Amplification Artifacts of the Fungal Barcode Marker in a Hybrid Mushroom.

Front Microbiol

Institute of Plant and Environment Protection, Beijing Academy of Agriculture and Forestry Sciences, Beijing Engineering Research Center for Edible Mushroom, Beijing, China.

Published: November 2019

AI Article Synopsis

Article 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 . Our results showed that PCR using the Phusion High-Fidelity DNA Polymerase has a per nucleotide error rate of about 4 × 10 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 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.

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Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6877668PMC
http://dx.doi.org/10.3389/fmicb.2019.02686DOI Listing

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