A simple PCR-based fluorometric system for detection of mutant fusion DNAs using a quencher-free fluorescent DNA probe and graphene oxide

Abstract

We propose a facile fluorometric system for detection of gene mutations using graphene oxide (GO). A fluorescent probe DNA anneals to a specific mutant gene and is degraded by the 5'→ 3' exonuclease activity of Taq polymerase during PCR, and the released fluorophore retains fluorescence after addition of GO without quenching.

Conventional PCR assisted single-component assembly of spherical nucleic acids for simple colorimetric detection of SARS-CoV-2

“…2 A . In the presence of target template, the pre-formed duplex of the linker and the target DNA, in the region between the two pecific PCR primers of the E gene amplicon, subjected to 5′- exonuclease activity of DNA polymerase [ 43 , 44 ]. As PCR proceeds through the region where the linker is annealed, the linker DNA is cleaved and removed from its partner during the amplification reaction.…”

Highly sensitive and specific screening of EGFR mutation using a PNA microarray-based fluorometric assay based on rolling circle amplification and graphene oxide

“…GO can also quench the uorescence of the uorophore-labeled single-stranded oligonucleotides via long-range energy transfer from the uorophore to the p-system of the GO molecule, 50 and the quenching efficiency is direct proportional to the ratio of GO to uorophore. 51,52 To achieve the optimal signal to background ratio, titration experiments of 1 mM F-DP with various concentrations of GO were performed, and the uorescence intensities of the resulting F-DP/GO solutions at 664 nm were recorded, respectively. As shown in Fig.…”

“…The single-stranded RCA product (RCAP) was then sensitively detected with uorescently labeled detection probe (F-DP) and graphene oxide (GO) system, which is an efficient nucleic acids sensing platform based on the different uorescence quenching effect of GO to uorescently labeled single-and double-stranded nucleic acids. [49][50][51][52][53][54] This method can achieve detection at femtomolar concentrations and ultrahigh mutation discrimination against EGFR wildtype. Capturing EGFR sequences using short (11 nt) PNA probe allows the use of unpuried samples and it offers an extremely simple and reliable way to prole EGFR mutations without the need of chemical modication for proling.…”

Graphene Oxide-Based Suppression of Nonspecificity in Loop-Mediated Isothermal Amplification Enabling the Sensitive Detection of Cyclooxygenase-2 mRNA in Colorectal Cancer

“…. On the other hand, GO has also been used to adsorb the primers and quench the background fluorescence from DNA staining dye in the PCR system to enlarge the difference between the target signal and the background and improve the specificity of PCR. ,− Furthermore, GO can also suppress nonspecific amplification caused by excess polymerase. , …”