pathological CAG repeat expansion [16], have been bred with repeat sizes up to around 500 CAG repeats [17]. Surprisingly, it was found that mice carrying the super-long repeat had a delayed onset of the disease phenotype and prolonged survival. The survival data showed a pronounced turning point at around 300 CAG repeats, indicating a fundamental change in phenotype. A concurrent study found a similar turning point [18]. Both studies showed a shift from predominantly intranuclear to predominantly cytoplasmic inclusions, suggesting that above a certain length the mutant protein cannot access the nucleus. Furthermore, the latter study reported that in mice with more than about 335 CAG repeats there was a .60% reduction in both mRNA levels for the transgene and expression of mutant protein compared with mice with 150 CAG repeats, suggesting that transcription of the gene was being inhibited. Significantly, a previous study has shown that HD homologue (Hdh) mRNA levels in knock-in mice containing 150 CAG repeats were already reduced compared with wild type, suggesting that a reduction in transcription becomes apparent even below the 300 CAG ‘turning point’ [19]. Nevertheless, it is possible that both a reduction in mRNA expression and nuclear entry of the mutant protein could contribute to the delayed phenotype in mice with longer repeats. In this study we set out to test the hypothesis that the observed change in phenotype of the R6/2 mouse involves the generation of unusual, non-B-form DNA at super-long CAG repeat lengths. There is already considerable evidence that the various triplet repeats generate unusual DNA structures, such as triplexes, hairpins forming in one or both strands of the DNA, slipped- strand DNA, and G-quadruplexes [20]. These structures have been detected through their effects on the mobilities of DNA on agarose gels (e.g. [21]), and have also been directly observed by electron microscopy (EM; [22,23]). Significantly, EM analysis requires extensive sample preparation and the imaging of the sample in vacuo, processes that result in the loss of some structural information. In contrast, atomic force microscopy (AFM) is able to image DNA at the single molecule level with only minimal sample preparation [24,25], revealing features that are not detectable using EM imaging. For instance, we have previously used AFM to visualize G-quadruplexes in DNA containing a G-rich fragment of the mouse Sc3 immunoglobulin switch region [26]. We found that the G-loops are asymmetric, and we showed directly that the G- rich strand forms a condensed structure, with a height appropriate to a four-stranded structure. This is in stark contrast to the extended single-stranded regions seen in the same DNA by EM [27], which are not consistent with the presence of G- quadruplexes in the form visualized. AFM images of triplet repeat DNA have been published before [28]; however, to the best of our knowledge, a systematic analysis of the structural features appearing at different repeat lengths has not been undertaken, particularly at super-long repeat lengths. Here we have used AFM to image DNA generated by PCR using genomic DNA isolated from tail snips taken from R6/2 mice as a template. We show that PCR-generated DNA with super-long repeats contains a variety of unusual structures, including hairpin loops and loop-mediated gross malformations, which, if they exist in vivo, would likely contribute to abnormal transcriptional regulation of the gene. Materials and Methods Generation of DNA DNA samples containing various CAG repeat lengths were generated by PCR from tail snips of either wild type or R6/2 mice [16], which carry the first exon of the human HD gene with a pathologically expanded repeat. The repeat length in the R6/2 Unusual Structures in Super-Long CAG Repeat DNA mice was originally around 140–150 CAG, but cohorts of mice have been generated with repeat lengths up to 485 [17]. Mouse husbandry was as described previously [29]. In some experiments, the forward and reverse PCR primers were tagged with biotin and DIG, respectively. PCR was carried out essentially as described in [11] with minor modifications. 35 cycles of PCR were carried out, using Taq polymerase (Applied Biosystems), with an annealing temperature of 65uC. The resulting PCR products from the R6/2 mice have 54 bp (59) and 77 bp (39) sequences flanking the CAG repeat core. Note that the GeneScan profiles of the PCR products revealed a range of sizes, and the peak sizes were used in our analyses. The quoted repeat lengths are therefore not absolute, and likely carry an error of 1–2%. Note also that the CAG repeat number measured by GeneMapper differs from that measured by sequencing. To convert the CAG repeat numbers determined by the GeneMapper technique to the CAG repeat number determined by sequencing technique (which more closely represents the true CAG repeat number in both HD transgenic mice and HD patients) the following formula was applied (Li and Whang, unpublished): True CAG repeat number = 1.0425 x GeneMapper CAG repeat number +1.2088. Where appropriate, DNA was purified using a DNA purifica- tion and concentrator kit (Zymo Research). Single-stranded (ss) DNA was PCR amplified from the human s-1 receptor gene in the pGH19 plasmid, kindly provided by Prof. M.B. Jackson (Department of Physiology, University of Wisconsin, Madison, USA). Primers were: forward, 59-TCCAAGCTT- GATGCAGTGGGCCGTGGGC-39; reverse, 59-TAAGCGG- CCGCAGGGTCCTGGCCAAAGAG-39. A 50-fold excess of forward primer was used, with 35 PCR cycles at an annealing temperature of 45uC, using Pfu Turbo DNA polymerase (Stratagene). Samples were purified using a QIAquick gel extraction kit. Enzyme Digestion Extensive digestion by the heat-resistant restriction enzyme TseI (New England BioLabs) was carried out for 60 min at 80uC in buffer containing 50 mM potassium acetate, 20 mM Tris-acetate, pH 7.9, 10 mM magnesium acetate and 1 mM dithiothreitol. The reaction was stopped by addition of 10% glycerol and 10 mM EDTA (final concentrations). Limited digestion was carried out at room temperature for 10 min, with one-fifth of the amount of enzyme used in the full digestion. Mung bean nuclease (New England BioLabs) digestion was carried out for 30 min at 30uC in buffer containing 50 mM sodium acetate, pH 5.0, 30 mM NaCl and 1 mM ZnSO4. Where appropriate, nuclease-treated DNA was melted by incubation at 94uC for 5 min. In this case, the nuclease was first inactivated by addition of 10% glycerol and 10 mM EDTA. EcoP15I (New England Biolabs) digestion was carried out for 1 h at 37uC with 0.03 U/ml of enzyme in buffer containing 100 mM NaCl, 50 mM Tris-HCl, pH 7.9, 10 mM MgCl2, 1 mM dithiothreitol and 1 mM ATP. At the end of the digestion the enzyme was inactivated by a 20-min incubation at 65uC. In all cases, DNA was purified using a QIAquick gel extraction kit prior to AFM imaging. Sample Preparation Disks of ruby muscovite mica (Agar Scientific) were attached to 13-mm steel pucks (Agar Scientific) using Loctite super-glue (Henkel Loctite Ltd.). The mica was cleaved with Scotch tape immediately prior to sample deposition to reveal an atomically flat surface. DNA was prepared at a concentration of ,0.04 ng/ml in imaging buffer (10 mM MgCl2, 10 mM Tris-Cl, pH 8.0). Droplets PLoS ONE | www.plosone.org 2 February 2011 | Volume 6 | Issue 2 | e17119