Journal: PLoS ONE
Article Title: A Degenerate Primer MOB Typing (DPMT) Method to Classify Gamma-Proteobacterial Plasmids in Clinical and Environmental Settings
Figure Lengend Snippet: DPMT validation for MOB F relaxases. A) Phylogenetic tree of MOB F relaxases. Triangles at the end of the branches represent a compressed group of very similar relaxases ( > 95%). A solid black arrow points to the prototype plasmid for each subfamily. Arrows point to plasmids that experimentally amplified, in spite of containing at least one mismatch in the 12 nucleotides of the CORE sequence. Relaxases contained in our reference collection ( Table 1 ) are denoted by an asterisk. Plasmids detectable by PBRT amplification  ,  ,  ,  ,  ,  ,  ,  ,  are underlined. New relaxase sequences uncovered by DPMT are shown in red. B) Alignment of the relaxase motifs used to design the MOB F degenerate primers. Colour code: red on yellow = invariant amino acids; blue on blue = strongly conserved; black on green = similar; green on white = weakly similar; black on white = not conserved. Black arrowheads point to the key residues that define the relaxase motifs. Different rectangles embrace the conserved amino acids used to infer the 3′ degenerate core of each oligonucleotide (F11-f, continuous black; F12-f, continuous dark grey; and F1-r, dashed black). C) Amplicons obtained with primers for subfamily MOB F11 (F11-f and F1-r). Lane 1, pSU1588; 2, pSU4280; 3, pSU10013; 4, pSU10014; 5, pSU10017; 6, pSU10018; 7, pSU10021; 8, pSU316; 9, pSU10022; 10, pSU10010; 11, R751; 12, pSU10028; 13, pSU10029; 14, pSU10056; 15, pSU10055; 16, pSU10001; 17, pSU10012; 18, pSU10011; 19, pSU10009; 20, pSU4601; 21, pSU10006; 22, pSU10007; 23, pSU10064; 24, pSU10059; 25, pSU10008; 26, pSU10039; 27, pSU10040; 28, pSU10041; 29, pSU10004; 30, pSU10003; 31, pSU10043; 32, pSU4830; 33, pSU10002; 34, negative control. Lane M, molecular mass marker, HyperLadder IV (Bioline). D) Amplicons obtained with primers for subfamily MOB F12 (F12-f and F1-r). Lanes as in (C).
Article Snippet: Amplicons were visualized after 2% agarose gel electrophoresis, using a GelDoc (BioRad Laboratories) and, when appropriate, sequenced by Macrogen Laboratories (Seoul, South Korea).
Techniques: Plasmid Preparation, Amplification, Sequencing, Negative Control, Marker