Estimation of Plasmid Lengths on ZEONEX®-Coated Cover Slips Using a Deposinator for Stretching of Single DNA Molecules

Abstract

Antibiotic resistance is one of the largest threats to global health, as it counters the way we treat bacterial infections. Antibiotic resistance genes can spread be-tween bacteria via plasmids. Simple and efficient methods for studying plasmids are thus important in the global fight against antibiotic resistance. This project optimizes and evaluates a novel molecular combing method for length estimation of plasmids – dragging a droplet of DNA solution across a ZEONEX®-coated cover slip followed by fluorescence microscopy. The method was optimized for pH, deposition speed and concentration using -DNA. A plasmid containing the resistance gene blaCTX−M−15 was linearized using either Cas9 or S1 nuclease, and the efficiency of both endonucleases was assessed. The plasmid was accurately estimated to 150 kbp by using -DNA as reference. The method shows promise, but further optimization is required to reduce noise that arise from broken DNA fragments.