title: Single base extension reaction-based surface enhanced Raman spectroscopy for DNA methylation assay.
abstract: DNA methylation is a key diagnostic maker for genetic disease, cancer progression and pharmcogenomics. So far various techniques have been developed for DNA methylation assay, but most of them are laborious and time-consuming. Here we develop a simple and highly sensitive DNA methylation assay based on single base extension reaction and surface enhanced Raman spectroscopy (SERS). In the presence of methylated DNA, gold nanoparticle-modified capture probe can couple with a cyanine 5-deoxyribonucleoside triphosphate (cy5-dGTP) through single base extension reaction, and generates a high SERS signal after further addition of gold nanoparticles to increase the local electromagnetic field. While in the presence of unmethylated DNA, gold nanoparticle-modified capture probe cannot couple with cy5-dGTP due to the presence of a mismatch base, and no SERS signal is observed. This single base extension reaction-based SERS can determine methylated DNA with a detection limit of 3 pM, and can even distinguish as low as 1% methylation level in tumor suppressor gene CDKN2/p16/MTS1 (p16) from the mixtures. Notably, the sensitivity of this assay has improved by 5 orders of magnitude as compared to reported gold nanoparticle-based colorimetric assay, and by 2 orders of magnitude as compared to microarray-based methylation-sensitive single nucleotide primer extension assay (Ms-SNuPE). This method might be further applied to detect the methylation status in tumor-linked genes for cancer diagnosis.
authors: Single-Molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China.
authors: Juan
authors: J
authors: Hu
authors: Chun-yang
authors: CY
authors: Zhang
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000235:genetics*; D019175:DNA Methylation; D008969:Molecular Sequence Data; D013059:Spectrum Analysis, Raman / Q000379:methods*
chemical_list: D004247:DNA