A UV Shadowing Technique Using a CCD Imaging System

To visualize DNA and RNA on a gel requires the use of a dye or a radiolabel. Traditionally, ethidium bromide, which allows DNA or RNA in gels to be visualized under UV light, has been the dye of choice for this application. However, because this stain is mutagenic and toxic, there has been an increase in the use of less toxic alternatives such as SYBR® Safe (Invitrogen, Carlsbad, CA) and GelRed™ (Biotium, Hayward, CA). Because these dyes can interfere with the structure of the nucleic acid, they have to be removed if the DNA in the gel needs to be used for further applications such as cloning. Therefore, scientists who want to extract DNA and RNA from their gels for reuse need a method that can visualize DNA and RNA without the use of any dyes.

One technique that can be utilized for visualizing nucleic acids without the use of dyes is UV shadowing,1,2 which is particularly effective for imaging oligonucleotides.3 The UV shadowing method described in this article requires transparent gels (i.e., polyacrylamide gels must be used) and employs short-wave UV light (254 nm) and a silica-coated thin-layer chromatography (TLC) plate. The gel is first placed on an acetate sheet or wrapped in clear plastic sheeting, and then on a silica-coated TLC plate; next, UV light is shone upon the gel. The nucleic acid absorbs light at this wavelength and casts a shadow against the fluorescent background, allowing the full-length nucleic acid to be imaged with a charge-coupled device (CCD)-based image analyzer.4 The amount of nucleic acid shadowed in a particular area of the gel is inversely proportional to the average pixel intensity in the corresponding image; therefore the amount of DNA or RNA can also be accurately quantified using a high-resolution CCD camera.

Method

Figure 1 - Method of setting up a gel for imaging by UV shadowing.

Figure 2 - G:BOX gel imaging system.

Oligonucleotides ranging in size from 3 to 13 bp were made up to 0.3 to 0.5 OD260 in 30 μL water. Formamide was added (15 μL); the solution was then vortexed briefly, heated to 80 ºC, and cooled rapidly in ice to denature the DNA. The oligonucleotides were loaded into a polyacrylamide gel (20% acrylamide/7 M urea). One lane was loaded with 2 μL of gel loading buffers (0.1% [w/v] solution of bromophenol blue) and 0.1% [w/v] xylene cyanol in 50% formamide in the marker lane. Since these loading buffers can sometimes appear as dark bands during UV shadowing, they are run alone in order to distinguish between the dyes and the shadow caused by the nucleic acids. The gel was run for 3 hr at a constant power of 20 W. It was wrapped with a piece of clear plastic sheeting, which was then laid onto a 20 × 20 cm silica gel-coated F60 254 TLC plate (Merck Chemicals Ltd., Nottingham, U.K.), as shown in Figure 1. The TLC plate, acetate, and gel were placed inside a G:BOX image analyzer (Syngene, Cambridge, U.K.) (Figure 2).

The G:BOX system was programmed to image the gel with overhead UV (254 nm) lighting shone directly onto the TLC plate; a UV shortpass filter was used to enable imaging of emitted fluorescence from the TLC plate, and thus allow imaging of any UV shadows. The gel image was then captured using Syngene GeneSnap software. The UV shadows were analyzed with Syngene GeneTools software to automatically calculate the size and molecular weight of the DNA in the bands.

Figure 3 - UV shadowing image showing oligonucleotides ranging in size from 3 to 17 bp (image kindly provided by the University of Southampton, U.K.), generated using a G:BOX CCD-based imaging system.

Results

The DNA in the UV shadowed gels appears as black bands, while the TLC plate appears white; thus the bands can be easily distinguished (Figure 3). The detection limit of the UV shadowing is approximately 0.3 µg of nucleic acid, and the quantification corresponds well to the amounts in the control oligonucleotide markers, indicating the accuracy of the technique. The largest band visualized using the technique was 300 bp; the smallest band was 2 bp long (figures not shown).

Discussion

The results demonstrate that UV shadowing can be both rapid and accurate using a sensitive CCD system. The G:BOX system is well suited for UV shadowing because its camera has megapixel resolution and a 16-bit gray scale; therefore it can accurately detect the gray scales in the image to produce an accurate account of DNA and RNA size and quantities in the gel. The camera is inside a darkroom fitted with an overhead UV (254 nm) light source that shines directly onto the TLC plate and has a filter wheel, which can be set with a UV shortpass filter to enable imaging of emitted fluorescence from the TLC plate. Because the darkroom is light-tight, the sample is protected from stray light, which could affect the imaging precision. The system is also supplied with GeneTools image analysis software, which analyzes the UV shadows to automatically calculate the molecular weight of the DNA or RNA in the bands.

The main benefit of using UV shadowing is that scientists can excise their DNA or RNA for further use without having to extract dye chemicals from the sample. This means that there will be less sample loss, time will be saved because there is no extraction step in the process, and the nucleic acid is more likely to remain in its native state than if a dye had been used to visualize it. Additionally, since a carcinogenic dye such as ethidium bromide is not needed, the associated biological and environmental hazards are reduced, as are the costs related to disposing of these dyes. Another benefit of UV shadowing is that it is less expensive than nontoxic dye. Although a TLC plate is required, it can be used multiple times because the plate is protected from damage by the sheet of plastic between the gel and the plate.

Conclusion

UV shadowing in conjunction with a high-resolution CCD-based system such as the G:BOX is a quick and accurate method for visualizing unlabeled nucleic acids, which will benefit scientists who need to use their DNA or RNA for further applications.

References

  1. Thurston, S.J.; Saffer, J.D. Ultraviolet shadowing nucleic acids on nylon membranes. Anal. Biochem. 1989, 178, 41–2.
  2. Hendry, P.; Hannan, G. Detection and quantitation of unlabeled nucleic acids in polyacrylamide gels. Biotechniques 1996, 20, 258–64.
  3. El-Sagheer, A.H.; Brown, T. Synthesis of the alkyne- and azide-modified oligonucleotides and their cyclization by the CuAAC (click) reaction. Curr. Protoc. Nucleic Acid Chem. 2008, 35, 4.33.1–21.
  4. Mahon, A.R.; MacDonald, J.H. et al. A CCD-based system for the detection of DNA in electrophoresis gels by UV absorption. Phys. Med. Biol. 1999, 44, 1529–41.

Dr. Bunn is Application Specialist, Syngene, Beacon House, Nuffield Rd., Cambridge CB4 1TF, U.K.; tel.: +44 (0) 1223 727100; fax: +44 (0) 1223 727101; e-mail: lindsey@syngene.com. Dr. El-Sagheer is Research Fellow, School of Chemistry, University of Southampton, Highfield, Southampton, U.K.

Comments