Unusual Structures in Super-Long CAG Repeat DNA Figure 2. Unusual structures occur in super-long CAG repeat DNA. (A) AFM image of wild type DNA with 8 CAG repeats (total length 298 bp). Scale bar, 500 nm. A colour-height scale is shown at the right. (B) DNA with 216 repeats (total length 779 bp). Scale bar, 500 nm. (C) DNA with 360 CAG repeats (total length 1211 bp). Scale bar, 500 nm. DNA fragments were classified into ‘normal and linear’ (inset, upper left; scale bar 100 nm), ‘convoluted’ (lower left; scale bar, 50 nm), ‘folded’ (upper right; scale bar, 50 nm), or ‘protruding’ (lower right, derived from another image of the same PCR product; scale bar 100 nm). (D) Gallery of zoomed images showing examples of convoluted DNA. (E) Graph showing the relationship between the percentage of DNA molecules with unusual structures and CAG repeat length. Numbers of DNA molecules analysed ranged from 1163 (114 repeats) to 203 (408 repeats). doi:10.1371/journal.pone.0017119.g002 Adhesion of the DNA to the mica surface for the purpose of AFM imaging was routinely achieved using 10 mM MgCl2 in the adsorption buffer. To exclude the possibility that this concentra- tion of Mg2+ was responsible for the generation of the unusual DNA structures in the DNA containing long CAG repeat lengths, in some experiments we coated the mica with poly-L-lysine before adsorption of the DNA in the absence of Mg2+. The positively charged poly-L-lysine coating binds DNA relatively strongly, so the DNA cannot equilibrate to its most energetically favourable two-dimensional conformation as it does in the presence of Mg2+. Consequently, we found that rather than being deposited in the relaxed conformation characteristically observed with the use of Mg2+ (Figure S1A), linear DNA (containing 360 CAG repeats) was deposited in serpentine conformations on poly-L-lysine (Figure S1B). However, this minor distorting tendency cannot account for the severe contortions observed in the figure: structures in which the DNA strand could not be traced from end to end were prevalent. Evidently, CAG repeat-induced conformational abnor- malities do not depend on the presence of Mg2+. The Unusual Structures do not Include Extensive Regions of ssDNA Given the repetitive sequence involved in these experiments, PCR synthesis in one direction might have been less effective than in the other, resulting in the generation of ssDNA. Other AFM PLoS ONE | www.plosone.org 5 February 2011 | Volume 6 | Issue 2 | e17119