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.
Highlights
The polymerase chain reaction (PCR), an in vitro method for the enzymatic replication of specific DNA sequences, enables the amplification of large amounts of target DNA fragments from one or a few template molecules (Mullis et al, 1986; Mullis and Faloona, 1987; Saiki et al, 1988)
While the application of thermostable DNA polymerases isolated from thermophiles such as Thermus aquaticus (Taq) have significantly improved the efficiency of PCR, artifacts can be produced during the PCR procedure (Cline et al, 1996; Ishii and Fukui, 2001; Kanagawa, 2003; Kurata et al, 2004; Acinas et al, 2005; Sharifian, 2010; Schloss et al, 2011; Bjørnsgaard Aas et al, 2017)
Our analyses revealed an overall nucleotide substitution rate of about 4 × 10−6 per nucleotide per replication using the Phusion R High-Fidelity DNA Polymerase following the recommended protocols to minimize mutations
Summary
The polymerase chain reaction (PCR), an in vitro method for the enzymatic replication of specific DNA sequences, enables the amplification of large amounts of target DNA fragments from one or a few template molecules (Mullis et al, 1986; Mullis and Faloona, 1987; Saiki et al, 1988). Due to its common use for bacterial taxonomy and environmental microbial ecology research, the 16S rRNA gene has been used as the template to estimate PCR error rate (Ishii and Fukui, 2001; Kurata et al, 2004; Acinas et al, 2005; Sharifian, 2010; Schloss et al, 2011) These studies have shown that template complexity can influence the frequencies of PCR artifacts (Farrelly et al, 1996; Suzuki and Giovannoni, 1996; Speksnijder et al, 2001; Sharifian, 2010; Schloss et al, 2011). The patterns of PCR artifacts based on intra-strain genetic variation of high copy number genes, especially those with potentially divergent intragenomic sequences, remain unknown
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
