Improving Nucleic Acid Gel Imaging

Nucleic acid gel imaging/documentation systems are a central tool that let you get answers quickly—what DNA fragments are present and/or absent? how big are they?— and then move on with your work. Using a gel imaging system should be fast and easy, with confidence in the reliability of the results. This article poses important points to consider when choosing a nucleic acid gel imaging system.

Stains, dyes, and light sources

Nucleic acids in a gel are visualized in two main ways: the more traditional stain ethidium bromide coupled with UV illumination, or the newer, safe nucleic acid dyes—such as SYBR— that fluoresce under blue LED light. The latter are also known as safe dyes, because they obviate exposure to hazardous UV light.

Dye compatibility is an important consideration, because an instrument should have the necessary optics to image current or planned dyes. Although researchers still seek instruments for UV illumination, there is increasing interest in safe dyes (or instruments that can do both). Besides posing a safety hazard for lab personnel, UV light can cause mutations in exposed DNA, which can be problematic for subsequent subcloning. “With safe dyes instead of UV illumination, you introduce fewer mutations, having significantly fewer issues with subcloning, and more colonies in the end,” says Mike Mortillaro, owner of Bulldog Bio.

Mortillaro says that Bulldog Bio’s instruments use a unique blue/green excitation range of safe dyes. “As compared with blue LEDs, this hits a sweet spot for both the green dyes and the red dyes for excitation, so you get very good excitation for both, as well as good signal and sensitivity,” he says. “In fact, you can get equivalent or better results with some of these safe dyes, so there's no reason any longer to work with UV illumination.”

Knowing what you need

While knowing the types of gels and perhaps blots you want to image is an important first step, researchers should not limit themselves, recommends April Wang, application scientist at Analytik Jena. Many instruments today offer both a UV transilluminator, and a blue LED light source for safe fluorescent dyes, as in Analytik Jena’s UVP GelStudio system. “The flexible excitation light and emission filter combinations allow researchers to perform more than just DNA gel imaging,” says Wang. “With the UVP GelStudio systems, researchers can image DNA gels, protein gels, and western blots.” Light source quality is also important, particularly its evenness and intensity. “The evenness ensures that the signal acquired on the edge of the sample area is comparable to the signal acquired at the center of the sample area,” she says. “Intensity is also a factor [because] stronger UV can help reduce the time needed for activation.”

LI-COR Biosciences also offers a range of systems with different light sources. The D-DiGit Gel Scanner is dedicated to nucleic acid imaging using safe dyes. For greater versatility, the Odyssey® platform includes fluorescence detection for western blotting, near-infrared illumination for detecting nucleic acids with a SYTO60 red fluorescent dye, and (in some models) ethidium bromide and other visible safe stains. In the near-infrared range of illumination, researchers are restricted to using SYTO60 for nucleic acids, “whereas when you get down into the blue-green region, there’s just a whole plethora of stains out there to choose from,” says Jeffrey Harford, senior product marketing manager at LI-COR Biosciences.

Sometimes researchers want an inexpensive nucleic acid imager because that’s all the functionality they need. Or they simply want to minimize the sharing of equipment or space — especially with the ongoing potential of coronavirus resurgence. Nucleic acid gel imagers are relatively inexpensive, but adding other capabilities like western blotting means a higher price tag. Incorporating additional functionalities will increase the versatility of a gel imaging system, but keep in mind that the additional optics required will cost more.

User-friendly software

Because most gel imagers rely on acquisition software for image capture, analysis, and/or storage, easy-to-use software is paramount. For example, Analytik Jena’s VisionWorks acquisition and analysis software has preset templates to capture images with one click. “VisionWorks allows researchers to create customized capture templates for different sample types,” says Wang. “Using the same software package to annotate, edit, and analyze a captured gel image helps researchers to streamline the analysis process and save time.”

Software with the ability to archive gel images, giving you easy and reliable access to specific images, can be especially valuable. “We’ve seen retractions of papers because researchers can't find their original data [when a reviewer asks to see it],” says Harford. “The ease-of-use of a system, and the ability to protect your future, is all tied into the software.” While some software runs the acquisition and annotation of gel images, not all software archives and stores the images for the long-term. “You want to have good software that you can count on, and if needed go back and look up data for a reviewer or publisher, or for a published article coming under scrutiny,” he says.

Optics

Generally, cameras in gel imaging systems are sufficient for capturing images of nucleic acid gels. But if available and/or financially feasible, enhanced camera optics—such as higher quality zoom lenses—can prove beneficial. “Nice optics help to maintain the depth of field for good imaging of even thicker gels, and across the entire plane of a larger gel, when you're looking at the XY coordinates,” says Mortillaro. “You want to make sure that everything is in focus and receiving the same level of light, so you can compare lanes on the edges with lanes in the middle.” Wang agrees that a sensitive camera with a wide aperture lens can produce higher quality images. “In addition, a camera with better dynamic range will allow researchers to see both the strong signal and weak signal on the same image,” she says.

Ongoing innovations in dyes and imaging will continue to improve tools for nucleic acid gel imaging. Mortillaro notes that dyes can alter the electrophoretic mobility of nucleic acids, making comparison to other results more challenging. “In new dyes, we look for a minimal or at least consistent electrophoretic effect, as well as stronger signals for the same size fragment in the same concentrations,” he says. “There are a lot of places you can look for improvement.”