A simple method for screening bacterial colonies for mutagenized sites in plasmid DNA

Abstract

Because of the multiple-step process that is involved in the detection of mutagenized restriction enzyme sites in plasmid DNA, a simple and accurate method was developed to analyse the plasmid DNA of site-directed mutagenesis experiments from bacterial colonies. The desired mutated part is located between the Eco RI restriction site on pUC19. Two mutagenic primers were designed to replace only one nucleotide on segments A and B of the bi-segmented genome of infectious bursal disease virus (IBDV). Two restriction sites were created for those mutations in each segment, Fsp I and Dra I, respectively. Following a protocol from the site-directed mutagenesis kit, the mutated plasmids were used to transform, and were propagated and maintained in DH5 alpha competent cells. Colonies were picked from the master plate, and used as DNA template for PCR. The PCR technique included the design of two pairs of primers, one for each segment, which were to amplify a region up to 1000 bp. Samples were pre-incubated for 3 min at 94 °C to induce bacterial lysis before starting the nucleic acid amplification. The PCR products 918 bp from segment A and 650 bp from segment B were digested with Fsp I and Dra I at 37 °C for 1 h. Products were resolved on 0.9% agarose gel which contained ethidium bromide. This method is simpler, faster and more accurate than the traditional method of mini-prep plasmid isolation and colony blot hybridization to identify the mutated plasmids.