Gomes-Pereira, M. and Monckton, D. G. (2017) Ethidium bromide modifies the agarose electrophoretic mobility of CAG•CTG alternative DNA structures generated by PCR. Frontiers in Cellular Neuroscience, 11, 153. (doi: 10.3389/fncel.2017.00153) (PMID:28611596) (PMCID:PMC5447772)
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Abstract
The abnormal expansion of unstable simple sequence DNA repeats can cause human disease through a variety of mechanisms, including gene loss-of-function, toxic gain-of-function of the encoded protein and toxicity of the repeat-containing RNA transcript. Disease-associated unstable DNA repeats display unusual biophysical properties, including the ability to adopt non-B-DNA structures. CAG•CTG trinucleotide sequences, in particular, have been most extensively studied and they can fold into slipped-stranded DNA structures, which have been proposed as mutation intermediates in repeat size expansion. Here, we describe a simple assay to detect unusual DNA structures generated by PCR amplification, based on their slow electrophoretic migration in agarose and on the effects of ethidium bromide on the mobility of structural isoforms through agarose gels. Notably, the inclusion of ethidium bromide in agarose gels and running buffer eliminates the detection of additional slow-migrating DNA species, which are detected in the absence of the intercalating dye and may be incorrectly classified as mutant alleles with larger than actual expansion sizes. Denaturing and re-annealing experiments confirmed the slipped-stranded nature of the additional DNA species observed in agarose gels. Thus, we have shown that genuine non-B-DNA conformations are generated during standard PCR amplification of CAG•CTG sequences and detected by agarose gel electrophoresis. In contrast, ethidium bromide does not change the multi-band electrophoretic profiles of repeat-containing PCR products through native polyacrylamide gels. These data have implications for the analysis of trinucleotide repeat DNA and possibly other types of unstable repetitive DNA sequences by standard agarose gel electrophoresis in diagnostic and research protocols. We suggest that proper sizing of CAG•CTG PCR products in agarose gels should be performed in the presence of ethidium bromide.
| Item Type: | Articles |
|---|---|
| Status: | Published |
| Refereed: | Yes |
| Glasgow Author(s) Enlighten ID: | Monckton, Professor Darren |
| Authors: | Gomes-Pereira, M., and Monckton, D. G. |
| College/School: | College of Medical Veterinary and Life Sciences > School of Molecular Biosciences |
| Journal Name: | Frontiers in Cellular Neuroscience |
| Publisher: | Frontiers Research Foundation |
| ISSN: | 1662-5102 |
| ISSN (Online): | 1662-5102 |
| Copyright Holders: | Copyright © 2017 Gomes-Pereira and Monckton |
| First Published: | First published in Frontiers in Cellular Neuroscience 11:153 |
| Publisher Policy: | Reproduced under a Creative Commons License |
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