Unusual Structures in Super-Long CAG Repeat DNA Figure 1. CAG repeat length affects PCR efficiency and accuracy. (A) GeneScan profiles of PCR products containing various CAG repeat lengths, showing that peak heights fall and peak widths rise as CAG repeat length rises. (Note the changing values on the y-axes). The red peaks indicate the positions of size markers (bp). A bp-scale is shown at the top. (B) GeneScan profiles of PCR products from two double mutant mice, showing that in both cases the longer repeat is under-represented. (C) Agarose gel of PCR products containing various repeat lengths. CAG repeat lengths are indicated above each lane. The left-hand side shows PCR products generated from DNA taken from mice carrying single copy of the transgene of varying sizes; the right-hand side shows PCR products from reactions containing equal amounts of the DNA from two of the mice shown on the left hand side; one carrying a CAG repeat of 111 and one other carrying a longer repeat. Note that the PCR reaction in the lane labelled 373 has failed. doi:10.1371/journal.pone.0017119.g001 216 CAG repeats (total length 779 bp, including flanking sequences) also appeared predominantly ‘normal’ and linear, with an average length of 240614 nm (n = 100), close to the expected 260 nm (Figure 2B). In contrast, when the DNA contained 360 CAG repeats (total length 1211 bp), various DNA structures became apparent (Figure 2C; see also Figure 3C for an image of 585-repeat DNA). Specifically, some of the DNA still appeared normal and linear (inset, upper left), but unusual structures were also present, which we term ‘convoluted’ (lower left), ‘folded’ (upper right), or ‘protruding’ (lower right, derived from another image of the same sample). A gallery of zoomed images of convoluted DNA structures is shown in Figure 2D. We imaged DNA samples of various CAG repeat lengths and analysed multiple images to produce a distribution of DNA forms for each sample. There was a progressive increase in the percentage of each type of unusual structure as the CAG repeat length increased, with over 50% of the molecules appearing unusual at the highest repeat length analysed (408 CAGs; Figure 2E). The measured length of the linear molecules in the 360-repeat sample was 370626 nm (n = 50), close to the expected 404 nm. It was not obvious from simple inspection whether the more complicated structures seen at longer repeat lengths consisted of single or multiple DNA molecules. To address this question, we measured the volumes of a number of linear molecules and a similar number of unusual structures in the sample containing DNA with 360 CAG repeats. The volumes determined (7526133 nm3 (n = 88) for the linear population and 8026158 nm3 (n = 72) for the unusual population) were not significantly different, indicating that the vast majority of the unusual structures consisted of single DNA molecules. PLoS ONE | www.plosone.org 4 February 2011 | Volume 6 | Issue 2 | e17119