Multicolor super-resolution DNA imaging for genetic analysis

Abstract

Many types of cancer and neurodegenerative diseases are caused by abnormalities and variations in the genome. We have designed a high-resolution imaging technique with high throughput and low cost for determining structural variations of genes related to genetic diseases. We initially mapped all seven nicking sites of Nb.BbvCI endonuclease enzyme on lambda DNA. Then we resolved densely labeled patterns of 107 nicking sites on human BAC DNA that is digested by Nb.BsmI and Nb.BbvCI endonuclease enzymes. This high density resulted in several dyes being closer together than the diffraction limit. Overall, detailed DNA nicking sites mapping with 100 bp resolution was achieved, which has the potential to reveal information about genetic variance and to facilitate medical diagnosis of several genetic diseases.

Figure 1

(a) Diagram of a DNA molecule with 3 Cy3 dyes to test the SHRImP method (b) Histogram of SHRImP distances between Cy3 dyes shows that the 3 distances between Cy3 pairs are 27, 61, and 95 nm, which are in good agreement with expected distances. (c) Three step photobleaching of the sample.

Figure 2

(a) Diagram of a DNA molecule that has Cy5-Cy3-Cy3 dyes on it. (b) Combined histogram of SHREC (red) and SHRImP (blue) analysis indicates that SHRImP distance between two Cy3 dye molecules is 56 nm, and two SHREC distances between Cy5-Cy3 molecules is 34 and 88 nm.

Figure 3

Resolving the Lambda DNA nicking pattern. (a) Lambda DNA nicking pattern by the enzyme Nb.BbvCI. Green stars show the expected location of labeled nicked sites. Site-B has two dyes and Site-C has three dyes in diffraction limited spots (b) an image of Tamra labeled nicking sites on YOYO stained Lambda DNA backbone. (c) Histogram of non-SHRImP distances. Peaks are at 1.47, 3.27, and 4.27 μm, which are close to the expected distances. (d) Histogram of SHRImP distances for site-C that has three peaks at 101, 202 and 312 nm, which is in good agreement with the expected distances of 104, 209, and 313 nm. (e) Histogram of SHRImP distances for site-B which has a peak at 104 nm that is also close to the expected distance of 110 nm.

Figure 4

Two-color super-resolution imaging of DNA molecules. (a) Diffraction limited imaging of BAC DNA fragments. DNA backbone is shown in blue (YOYO-1 staining). Nb.BsmI nicking sites are in yellow and Nb.BbvCI sites are in red. DNA fragments are rotated to be aligned with all others based on the nicking pattern. (b) Image of a single DNA fragment (the fragment on panel a shown with an arrow). The image on the left is the superposition of the green and red images on the right. Excitations in the green and red channels are done alternatively. Arrows show SHRImP spots in each channel. (c) Gaussian profiles of spots in the green channel on the DNA fragment. Colors in the profiles shows varying intensities (blue to yellow is low to high intensities). The graph includes Gaussian profiles of the SHRImP spot (shown with arrow) before and after photobleaching (first and second graph). Third graph is the Gaussian profile of the second dye on the SHRImP spot. It is found by subtracting spot’s intensity profile after photobleaching from that of before photobleaching (d) Gaussian profiles of dyes on the red channel on the same DNA fragment. Spot shown with arrow is a SHRImP spot. Gaussian profiles of the spot with before and after photobleaching are shown in first two graphs. Third one is Gaussian profile of the second dye on the SHRImP spot. (e) Fiduciary markers, which are 100 nm in diameter and 1.5 μm apart from each other, are used to map the green channel onto red channel to perform SHREC analysis. Alternating images of the fiduciary marker are taken in green and red channels. Then, a mapping matrix is produced by localization of every spot on the illumination area according to the algorithm in Goshtasby et al.^,

Figure 5

BAC DNA mapping with two-color SHRImP analysis. (a) Locations of experimentally determined nicking sites on BAC DNA are shown above the histogram. DNA was nicked by Nb.BsmI at 71 locations (labeled by Tamra dyes) and by Nb.BbvCI at 35 locations (labeled by Cy5 dyes). The histogram is created with bin sizes of 200 nm. Experimentally located nicking sites for Tamra and Cy5 dyes are shown with green and red bars, respectively. (b) The histogram on the left shows the region from 19.5kb to 24.5kb in which four Tamra and three Cy5 labeled sites are resolved as expected. The histogram on the right has 11 nicking sites and covers an 11kb region from 44kb to 55kb. Five Tamra and six Cy5 labeled sites are resolved with two-color SHRImP in expected locations.