title: A simple microfluidic assay for the detection of ligation product.
abstract: We present a novel microfluidic-based approach to detect ligation products. The conformal specificity of ligases is used in various molecular assays to detect point mutations. Traditional methods of detecting ligation products include denaturing gel electrophoresis, sequence amplification, and melting curve analysis. Gel electrophoresis is a labor- and time-intensive process, while sequence amplification and melting curve analysis require instruments capable of accurate thermal ramping and sensitive optical detection. Microfluidics has been widely applied in genomics, proteomics, and cell cytometry to enable rapid and automated assays. We designed an assay that fluorogenically detects ligation products following a simple magnetic separation through a microfluidic channel. 100 nM of synthetic HIV-1 K103N minority mutant templates were successfully detected in 30 min. This simple and rapid method can be coupled with any ligation assay for the detection of ligation products.
authors.affiliation: Center for Biomedical Engineering, School of Engineering, Brown University, 182 Hope Street, Providence, RI, 02912, USA.
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.forename: Jingjing
authors.initials: J
authors.lastname: Wang
authors.forename: Johann
authors.initials: J
authors.lastname: Roebelen
authors.forename: Anubhav
authors.initials: A
authors.lastname: Tripathi
mesh_terms: D001681:Biological Assay; D015342:DNA Probes / Q000138:chemical synthesis / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D015497:HIV-1 / Q000235:genetics; D006801:Humans; D022521:Ligase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D008280:Magnetics / Q000295:instrumentation / Q000379:methods*; D044085:Microfluidics / Q000295:instrumentation / Q000379:methods*; D017354:Point Mutation; D012680:Sensitivity and Specificity
chemical_list: D015342:DNA Probes; D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes