Detection and Quantitation of DNA Mutations Based on Gold Magnetic Nanocomposites and Single Primer Extension Assay

摘要

A new method for DNA mutations detection and quantification was developed by using Fe3O4@SiO2@Au (Gold-coated magnetic nanocomposites, GMNPs) and single primer extension assay (SPEA). In this scheme, the single fluorescent-labeled primers were extended specifically with target DNA containing mutation sites from wild types, and subsequently hybridized with biotin-labeled probes captured by streptavidin-modified GMNPs, followed by detection of fluorescence signals. To test the feasibility of the method, we attempted to quantify the synthesized DNA segments from epidermal growth factor receptor (EGFR) with known mutant L858R and T790M under optimum conditions. The synthesized target DNA was relatively quantified by calculating the ratio of fluorescence intensity between the target and reference genes. Results exhibited an excellent readout due to the use of GMNPs with low fluorescence background, and a fine linear correlation, with target concentration ranging from 1.25 to 20 nM. This approach has potential in development of a simple, rapid, special and low-cost technique for DNA mutation quantification from real samples.