Journal: Frontiers in Cellular Neuroscience

Article Title: Ethidium Bromide Modifies The Agarose Electrophoretic Mobility of CAG•CTG Alternative DNA Structures Generated by PCR

doi: 10.3389/fncel.2017.00153

Figure Lengend Snippet: Effect of ethidium bromide on the mobility of CAG•CTG sequences in agarose gels. (A) The presence/absence of ethidium bromide modifies the mobility of PCR amplified expanded CAG•CTG repeats during agarose gel electrophoresis. PCR products containing 5, 22, 56 or 200 CAG•CTG repeats were individually isolated from the human DM1 locus by serial dilution, subsequently amplified with various combinations of DM-specific oligonucleotide primers (DM-A, DM-H, DM-C, DM-BR, DM-DR and DM-ER) and resolved through 1.8% (w/v) agarose gels, with (left) or without (right) 500 nM ethidium bromide in both the gel and 1X TBE running buffer. The amplified products were subsequently detected by Southern “squash” blot hybridization. The autoradiographs illustrate the increased mobility of alternative expanded CAG•CTG conformers in agarose gels in the presence of ethidium bromide. Note the multiple alternative products observed in the absence of the intercalating chemical. The scale on the left represents the position and sizes of the molecular weight markers in bp (M). (B) The absence of ethidium bromide in the gel and running buffer results in the detection of additional slow migrating species during agarose gel electrophoresis of expanded CAG•CTG sequences. The autoradiograph shows a close-up of DM1 PCR products resolved in an agarose gel in the absence of ethidium bromide. Note the position of the putative B-DNA structures (1) and non-B-DNA structures (2 and 3). (C) The intense fast-migrating bands observed in the absence of ethidium bromide exhibited the same electrophoretic mobility as the single band detected when ethidium bromide was present. The ratio between the relative mobility in agarose gel and the actual size (bp apparent /bp actual ) of the DNA species is shown for PCR products containing 22, 56 and 200 repeats. The graph shows a significant difference between the electrophoretic mobility of the slow migrating DNA species detected in the absence of ethidium bromide and the single band detected in the presence of the dye (* p < 0.05; *** p < 0.001; one-way analysis of variance (ANOVA)). (D) Pre-incubation with ethidium bromide fails to eliminate additional slow migrating species during agarose gel electrophoresis of PCR-amplified expanded CAG•CTG repeats. DM1 alleles with different repeat numbers were amplified by PCR using multiple oligonucleotide primer combinations, indicated in the figure above each lane. Half of each PCR product was incubated with 500 nM of ethidium bromide for over 48 h prior to electrophoresis and hybridization analysis. The autoradiographs do not reveal any detectable difference in the electrophoretic profile between treated and non-treated samples. (E) Ethidium bromide modifies the agarose electrophoretic mobility of CAG•CTG products generated by bulk PCR amplification of 20–100 ng of template genomic DNA. Ethidium bromide post-staining revealed slow migrating DNA species containing 56 and 200 CAG•CTG repeats, which were undetected when the dye was added to the gel and running buffer.

Article Snippet: Non-denaturing polyacrylamide gel electrophoresis (PAGE) was carried out in a BioRad Protean II gel apparatus, using 8% (w/v) non-denaturing acrylamide/bis (29:1) gels, containing 10% (v/v) glycerol, in 1X TBE (90 mM Trizma base, 90 mM orthoboric acid, 2 mM EDTA).

Techniques: Amplification, Agarose Gel Electrophoresis, Isolation, Serial Dilution, Hybridization, Molecular Weight, Autoradiography, Incubation, Electrophoresis, Generated, Staining