Progress in whole-genome sequencing using short-read (e.g., <150 bp), next-generation sequencing technologies has reinvigorated interest in high-resolution physical mapping to fill technical gaps that are not well addressed by sequencing. Here, we report two technical advances in DNA nanotechnology and single-molecule genomics: (1) we describe a labeling technique (CRISPR-Cas9 nanoparticles) for high-speed AFM-based physical mapping of DNA and (2) the first successful demonstration of using DVD optics to image DNA molecules with high-speed AFM. As a proof of principle, we used this new “nanomapping” method to detect and map precisely BCL2–IGH translocations present in lymph node biopsies of follicular lymphoma patents. This HS-AFM “nanomapping” technique can be complementary to both sequencing and other physical mapping approaches.

Follow this and additional works at: http://scholarscompass.vcu.edu/phys_pubs Part of the Physics Commons Reshchikov, M.A., Olsen, A.J., Bishop, M.F., et al. Superlinear increase of photoluminescence with excitation intensity in Zn-doped GaN. Physical Review B, 88, 075204 (2013).We have observed a superlinear increase of photoluminescence (PL) intensity in a narrow range of excitation intensities for Zn-doped GaN. The characteristic intensity at which the abrupt increase occurs increases with increasing temperature. This is unlike the usual observations for defects in semiconductors in which the PL intensity increases linearly with excitation intensity, saturating at high intensity because defects become saturated with photogenerated charge carriers. The observed phenomenon is attributed to a redirection of electron and hole flow from nonradiative centers at low excitation intensity to a recombination path via the Zn Ga acceptor at high excitation intensity. This is the same explanation responsible for the abrupt thermal quenching of PL reported earlier [Reshchikov et al., Phys. Rev. B 84, 075212 (2011).]

Quantitative polymerase chain reaction is the current “golden standard” for quantification of nucleic acids; however, its utility is constrained by an inability to easily and reliably detect multiple targets in a single reaction. We have successfully overcome this problem with a novel combination of two widely used approaches: target-specific multiplex amplification with 15 cycles of polymerase chain reaction (PCR), followed by single-molecule detection of amplicons with atomic force microscopy (AFM). In test experiments comparing the relative expression of ten transcripts in two different human total RNA samples, we find good agreement between our single reaction, multiplexed PCR/AFM data, and data from 20 individual singleplex quantitative PCR reactions. This technique can be applied to virtually any analytical problem requiring sensitive measurement concentrations of multiple nucleic acid targets.

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AbstractThis paper presents a strategy for an unsupervised workflow for identifying epithelial cells in microscopic images and characterizing their morphological and/or optical properties. The proposed method can be used on cells that have been stained with fluorescent dyes and imaged using conventional optical microscopes. The workflow was tested on cell populations that were imaged directly on touch/contact surfaces and stained with nucleic acid dyes to visualize genetic content. Our results show that this approach could be a useful strategy for characterizing differences in staining efficiency and/or morphological properties of individual cells or aggregate populations within a biological sample. Further, they can potentially reduce the laborious nature of microscopic analysis and increase throughput and reproducibility of similar studies.