Unusual Structures in Super-Long CAG Repeat DNA Figure 3. Convoluted DNA does not contain extensive regions of ssDNA. (A) AFM image of human s-1 receptor DNA, generated by PCR using a 50-fold excess of forward primer. Two types of structure can be seen: 690-bp dsDNA, which is linear and ‘normal’, and ssDNA, which is very dense and compacted. Scale bar, 250 nm. (B) s-1 receptor DNA after extensive Tse1 digestion at 80uC. The enzyme has cleaved the dsDNA, leaving shorter fragments, but the ssDNA (arrows) remains unaffected. Scale bar, 250 nm. (C) DNA containing 585 CAG repeats. Note that some molecules are linear, whereas others are folded or convoluted. Scale bar, 500 nm. (D) 585-repeat DNA after extensive Tse1 digestion at 80uC. A dense homogenous ‘background’ of short (,6-bp) fragments remains, together with a few aggregations of these fragments, and one fragment of ,60 bp, which likely represents a flanking region. Scale bar, 250 nm. (E) Undigested 585-repeat DNA. Scale bar, 500 nm. (F) 560-repeat DNA after limited Tse1 digestion at room temperature. The enzyme has preferentially cleaved linear regions of DNA, leaving behind the anomalous regions. Scale bar, 250 nm. doi:10.1371/journal.pone.0017119.g003 studies (e.g. [35]) have shown that under some conditions ssDNA forms convoluted structures visibly distinguishable from dsDNA, which is characteristically linear and uncontorted. We therefore tested whether the longer repeat DNA molecules contained extensive regions of ssDNA. To do this, we used the restriction enzyme TseI, which cuts at the sequence 59-GC(A/T)GC-39. To remove any intra-stand base pairing that would exist in ss CAG repeat DNA, incubations with the enzyme were carried out at 80uC; conveniently, TseI is stable at this temperature. In an initial experiment we deliberately generated ssDNA by amplifying a test sequence (the human s-1 receptor sequence; [36]), using PCR with a 50-fold excess of the forward primer. This reaction generated both 690-bp dsDNA and the corresponding ssDNA. In AFM images, the two populations were clearly distinguishable (Figure 3A). The dsDNA looked normal and linear, and identical to dsDNA generated using the same template but with equal concentrations of the two primers (Figure S2A). In contrast, the ssDNA was highly compacted, likely as a result of intra-stand base pairing. After an extensive TseI digestion at 80uC, the ssDNA subpopulation was unaffected (arrows in Figure 3B), whereas the dsDNA was digested into fragments of various sizes, as expected given that the s-1 receptor sequence contains five TseI sites. s-1 PLoS ONE | www.plosone.org 6 February 2011 | Volume 6 | Issue 2 | e17119