Fluorescent Detection and Isolation of DNA Variants Using Stabilized RecA-Coated Oligonucleotides

Shown in the table are results from an adaptation of our capture method that is capable of isolating sequences that differ by as little as 1 nt. The first two columns from the left describe five different plasmid targets that were used and the starting proportion of Kan^cnv sequence in the sample: two plasmids that differ by only a single nucleotide, (1) pWE15 Kan^R carrying a wild-type kanamycin gene (TAT codon): (2) pWE15 Kan⁵ carrying a mutant kanamycin gene (TAG codon); and three plasmid samples with a mixture of plasmids carrying a mutant kanamycin gene TAG codon) and a conservative replacement (Kan^cnv) that confers the Kan^R phenotype (TAC codon)—(3) pWE15(CNVB1), (4) pWE15(CNVD3), (5) pWE15(CNVD5). Two different preparations of these plasmid samples were used as templates for the capture assay, either supercoiled or topoisomerase I relaxed cccDNA. In each of these plasmid forms, results are presented before separation (Colonies presep) and after the biotin-labeled complexes were separated by binding to strepavidin-conjugated paramagnetic beads (Colonies postsep) for each preparation. The numbers presented are actual colony numbers obtained after transforming equal samples of each condition into bacteria and replica plating for kanamycin (Kan) and ampicillin (Amp) LB plates and the proportion of Kan to Amp (% Kan CNV).