DNA digestion and ligation on a microfluidic chip for rapid gene engineering

Abstract

A microfluidic system for fluorescence detection on digested and ligated DNA fragments was developed as one of the development for rapid genetic engineering. This system was integrated onto a silicon chip with enzymatic reaction taken place within the functionalized chip thereafter. Functionalized chip technique was a key process for better binding affinity between the chip surface and the DNA. By making use of fluorescent dye, measurement of splicing and attachments segments was possible. This technique will allow a DNA restriction map to be construe using an automated system by optical mapping quickly.Stepwise functionalizing of chip with APTES, biotin and avidin were completed within 4 hours. Biotin-synthesize double stranded DNA (dsDNA) labeled with FAM and CY5 at each ends respectively was immobilized onto the surface of the chip within 1 hour. Digestion by BamH1 and ligation by DNA T4 ligase were both favorable at 5 minutes. Compared this micro-system to conventional molecular diagnostic, which will took 4 hours for digestion and 12 hours for ligation, such system had its advantages over time and sample volume containment.