Transient stem-loop structure of nucleic acid template may interfere with polymerase chain reaction through endonuclease activity of Taq DNA polymerase

As a standard molecular biology technique, PCR uses DNA polymerase to detect, amplify and manipulate DNA targets. Due to its effect of exponential amplification, PCR can achieve high sensitivity required for detecting targets of low abundance. Therefore, it has become the method of choice for the majority of nucleic acid-based tests. In PCR reactions, DNA templates are first unwound into single strands, followed by a quick temperature drop when transient intramolecular secondary structures may form first within the single-stranded templates due to reaction kinetics. In this study, we showed that the adverse effects of stem-loop structures on PCR performance were directly correlated with their thermal stability. Moreover, fractions of intermediate PCR products of templates with stable stem-loop structures were significantly shorter than those without. It was further demonstrated that when encountering the duplex region of such a structure during the PCR extension step, the endonuclease activity of Taq DNA polymerase mediated by its 5'-3' exonuclease activity could digest template strand, resulting in stem-loop structure unwinding and subsequent completion of replication to produce truncated products. This work thus provided some new mechanistic insights into the complex nature of PCR assays, a frequently encountered but neglected aspect of this widely used technique.