Multiplex genotyping of SNPs in genomic DNA via hydrogel-based assay mediated with MutS and polyethylene glycol

In this study, a hydrogel-based multiplex SNP genotyping platform specifically designed for genomic DNA is presented. This platform integrates the ligation detection reaction (LDR) and rolling circle amplification (RCA) techniques within a hydrogel-based multiplex sensing system, enabling adaptable and sensitive SNP genotyping for genomic DNA. To enhance the specificity of the assay, MutS protein and polyethylene glycol are introduced into the protocol, reducing the non-specific ligation and RCA reactions synergistically. With significant specificity improvement of over 10-fold, three types of SNPs within an artificially constructed ∼1000 bp double-stranded DNA (dsDNA) are successfully genotyped with double-digit picomolar sensitivity. Furthermore, the practical applicability of the developed process for the origin identification of raw materials is demonstrated by genotyping three types of SNPs within genomic DNA obtained from two closely related plant species, Korean ginseng (Panax ginseng) and American ginseng (Panax quinquefolius), containing ca. 3.5 gigabase genome size. Of notable significance, this study marks the premiere achievement in PCR-free multiplex genotyping of SNPs in genomic DNA using a single fluorophore.PMID:37714061 | DOI:10.1016/j.bios.2023.115670
Source: Biosensors and Bioelectronics - Category: Biotechnology Authors: Source Type: research