Abstract
Aiming at improved specificity, nanoparticle assisted polymerase chain reaction (PCR) has been widely studied and shown to improve PCR. However, the reliability and mechanism of this method are still controversial. Here, we demonstrated that 1 μg/mL of graphene oxide (GO) effectively enhances the specificity of the error-prone multi-round PCR. Mismatched primers were designed as interference to produce nonspecific products when the same amounts of matched and mismatched primers were added into semi-multiplex PCR. It was found that GO can enhance specificity by suppressing the amplification of mismatched primers. We monitored the primer-template-polymerase-GO interactions involved in the PCR using a capillary electrophoresis/laser-induced fluorescence polarization (CE-LIFP) assay. The results showed that the addition of GO promoted the formation of a matched primer-template complex, but suppressed the formation of a mismatched primer-template complex during PCR, suggesting that interactions between the primers and GO play an essential role. Furthermore, we successfully amplified the FOXL2 gene from PEGFP-N1 vectors using GO to eliminate the nonspecific products in PCR. Taken together, these results suggest that the GO can be used as an efficient additive for improving the conventional PCR system.
Highlights
Polymerase chain reaction (PCR) amplifies a specific region of a DNA strand to generate thousands to millions of copies of a particular DNA sequence
We found that graphene oxide (GO) significantly improved polymerase chain reaction (PCR) specificity at appropriate concentrations
We demonstrated that in the presence of GO, the nonspecific products formed by mismatch primers can be suppressed in PCR
Summary
Polymerase chain reaction (PCR) amplifies a specific region of a DNA strand to generate thousands to millions of copies of a particular DNA sequence. PCR has become one of the most important techniques in modern biological and medical science It has a variety of applications, including DNA cloning for sequencing, functional analysis of genes, diagnosis of hereditary and infectious diseases, and identification of genetic fingerprints[1,2,3,4,5,6,7,8,9,10]. Gold nanoparticles and reduced graphene oxide (RGO) were recently reported to reduce nonspecific fragment formation in multiple-round PCR16–18. This enhancement effect of nanoparticles on PCR specificity might be attributable to two potential mechanisms: selective binding to single-stranded DNA (ssDNA) in a manner analogous to SSB16 and heat transfer enhancement by the superior energy transport properties of nanoparticles[17,19]. We further confirmed that GO promoted matched primer-template complex formation and suppressed www.nature.com/scientificreports/
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