(45 ml) were applied to the freshly-cleaved mica, and incubated at room temperature for 5–10 min. The surface was washed ten times with 1 ml biotechnology performance certified (BPC) water (Sigma-Aldrich). Excess liquid was removed using a gentle stream of water-filtered nitrogen, and samples were placed in a non- vacuum desiccator overnight. Where appropriate, mica was treated with 50 ml of 0.1% poly-L-lysine (Sigma-Aldrich) for 10 min followed by washing, before adsorption of DNA. AFM Imaging Imaging was performed using a Multimode Nanoscope IIIa atomic force microscope (Veeco Digital Instruments, Santa Barbara, CA, U.S.A.) fitted with an AS130 ‘J’ scanner. All samples were imaged in air using tapping mode. Micro Cantilever silicon tips with a spring constant of ,42 N/m (Olympus) driven at a resonant frequency of ,300 kHz were used as probes. The instrument was operated with Veeco’s Nanoscope software (v5.31r1) and Nanoscope IIIa interface (Digital Instruments). Integral gain was set to 0.2, proportional gain to 0.4, scan rate to 5 Hz, and z-limit to 1 mm. Images contained 5126512 pixels of height data, and image area was set between 1 mm and 4 mm, depending on DNA size and the density of particles on the surface. Colour was used to encode height. Scales were adjusted between experiments to present clear images of the DNA. These adjustments were necessary because of the variation in both tip performance and background quality between experiments. Image Analysis First order ‘flattening’ was applied to raw images in Nanoscope to compensate for the intrinsic bow resulting from the nature of the AFM’s piezo-actuated scanner. The height scale, colour contrast, and colour offset were set individually for each image to maximize clarity. (These settings do not alter fundamental image values). Particle volumes were assayed with the ‘grain analysis’ function in SPIP (Image Metrology). DNA length was measured using the ‘line plug-in’ running in ImageJ (1.37v, NIH). Force Measurement All procedures were carried out at room temperature. AFM cantilevers made of silicon nitride with a specified spring constant of 0.02 N/m (OMCL-TR400PSA; Olympus Co. Ltd., Japan), and freshly-cleaved mica sheets, were treated with 3-aminopropyl- triethoxysilane (APTES; Sigma) and N,N-diisopropylethylamine for 1 h. After washing with MilliQ water, cantilevers and mica were incubated with 1 mM glutaraldehyde for 10 min. After further washing in water, the cantilevers were dipped into streptavidin (0.1 mg/ml) in phosphate-buffered saline (PBS), and incubated for 20–30 min. The mica was treated with anti-DIG antibody (10 mg/ml) for 20–30 min. Cantilevers and mica were then washed with PBS, and with 50 mM KCl, 10 mM Tris-HCl buffer, pH 7.6. The cantilevers were dipped into the appropriate DNA solution (3–5 ng/ml) for 10 min. After washing in Tris/KCl buffer, cantilevers and mica were used immediately. Force measurements were performed with a Digital Instruments Multimode atomic force microscope equipped with an E-scanner and a Nanoscope IIIa controller with an in-line electronics extender module (Veeco Digital Instruments, Santa Barbara, CA, U.S.A.). Measurements were carried out in Tris/KCl buffer (above). The pulling speed was 100 nm/s, the pulling distance was 500 nm, and the trigger point was set at 50 nm. The data were analysed using SPIP. Rupture forces were obtained from force- extension curves. Unusual Structures in Super-Long CAG Repeat DNA Results Apparent Anomalies in Genotyping of Double Mutant Mice with Super-Long CAG Repeats This study was prompted by anomalies encountered in the routine genotyping of R6/2 mice by PCR, using genomic DNA isolated from tail snips as a template. It was found that as the CAG repeat length increased, PCR yields fell, and the PCR products became progressively less well defined, a finding that has been reported previously by others [30,31,32]. This phenomenon can be seen in GeneScan profiles (Figure 1A). This became problematic when genotyping mice with CAG repeats longer than 400, and even more so when we tried to measure the CAG repeat lengths from double mutant mice. Because mice with super- long CAG repeats are fertile, offspring carrying two copies of the transgene can be generated. However, matings between hemi- zygotic mice appeared to generate significantly fewer double mutant offspring than expected when the repeat length of the parents was long (350–500 CAG repeats), but not when it was short (250–260 CAG repeats). On those occasions when two peaks were seen, GeneScan profiles typically showed that the longer of the two repeats was under-represented and more diffuse (Figure 1B). More commonly, there was no second peak, although subsequent phenotyping and histological analysis [17] showed that the proportion of mice carrying two copies of the transgene was 0.25, as expected. The unequal amplification of differently sized alleles was recapitulated in vitro (Figure 1C). When PCR reactions were run using template DNA from mice carrying a single copy of the transgene (lanes 1–6 on the left hand side of the gel), PCR products became increasingly faint as the repeat length increased. The preferential PCR amplification of shorter template was shown in an experiment in which template was generated by in vitro mixing DNA from two different animals. When the template DNA from a mouse carrying a CAG repeat of 111 was mixed with that of a mouse carrying a longer CAG repeat (Figure 1C; right-hand side of the gel), the longer of the two PCR products was clearly under-represented or, with the longest repeat sizes, undetectable. This phenomenon was reported some time ago for CAG repeats in the human androgen receptor gene [33] and for GAA repeats in the Friedreich’s ataxia gene [34]. Interestingly, the former paper contained speculation that the HD locus might show the same effect, as we have now demonstrated. AFM Imaging Reveals Unusual Structures in DNA with Super-Long CAG Repeats A potential explanation of the results described above is that super-long DNA adopts unusual structures that reduce the efficiency of the PCR reaction. Such a phenomenon occurring in vivo might also account for the observed reduction in transcription of the gene encoding htt as repeat length entered the super-long range [18]. To test this idea, we analysed DNA samples of various CAG repeat lengths by AFM imaging. We found that the structural profile of the DNA changed significantly as repeat length increased (Figure 2). DNA generated from wild type mice, appeared as short linear molecules of length 9665 nm (S.D.; n = 100; Figure 2A). The PCR fragment generated from the region containing the (CAG)2CAA(CAG)4 sequence carried by wild type mice was expected to have a total length of 298 bp. We have shown in a previous AFM-based study of plasmid DNA [25] that an appropriate nm-to-bp conversion factor is 0.334 nm/bp. 298-bp molecules should therefore be 99 nm long, very close to the observed value. Note also that we have previously used AFM to image various DNA molecules that do not contain CAG repeats and have only ever seen linear structures (e.g. [24,25]). DNA with PLoS ONE | www.plosone.org 3 February 2011 | Volume 6 | Issue 2 | e17119