Agarose gel electrophoresis of DNA

This article discusses the basics of agarose gel electrophoresis, including how it works, how the equipment functions and its various applications. To follow this article, as basic understanding of the structure of DNA and proteins is helpful.

Contents

Introduction

Agarose gel electrophoresis is one of the most commonly performed life science laboratory techniques. Researchers use this technique to separate biological molecules based on their size. The ability to separate molecules by size can be useful in a range of research applications such as identifying unknown samples compared to known results or performing accuracy or quality control during other procedures.

Agarose gel electrophoresis is most commonly used in the separation of DNA molecules and so is frequently used during DNA manipulation techniques, or studies involving identifying individuals based on their unique DNA sequence. Below we discuss just some of the applications of agarose gel electrophoresis of DNA, how it works and what equipment is required to perform the technique.

How does it work?

Figure 1: The chemical structure of DNA.

DNA has a distinct chemical structure, in which the nucleobases, the letters of the DNA code, are joined by a backbone of a sugar, deoxyribose, and a phosphate group. This structure is shown in figure 1. As can be seen in the figure, the backbone of the DNA contains a negative charge for every nucleobase present, making the mass-to-charge ratio of DNA the same across different fragment sizes. Because of this negative charge, when we apply an electrical field to a solution containing DNA, the DNA molecules will migrate towards the positively charged electrode.

The Gel Matrix

In agarose gel electrophoresis we introduce a gel matrix, imagine several layers of sieves or netting, which the DNA migrates through along the voltage gradient towards the positive electrode. This matrix creates resistance and means that smaller molecules migrate more quickly while larger molecules migrate more slowly. The difference in migration rate is how we separate the different sizes of DNA molecule to determine their length. The gel matrix is created by dissolving a natural polysaccharide called agarose, derived from a type of seaweed, in a conductive buffer typically at around 1% agarose, and allowing it to set into a gel. The pore size in this gel matrix is well suited to the separation of DNA and the speed of migration can be influenced by increasing or decreasing the percentage of agarose in the mixture.

Running the gel

Figure 2: Running of an agarose electrophoresis gel. 1 – wells are formed using combs during casting. 2-4 samples are loaded with a pipette. 5-6 – Electrical field is applied to separate samples.

For a typical agarose gel electrophoresis procedure, the gel matrix is cast as a horizontal slab. Plastic combs are used to create indentations, or wells, into which the DNA is loaded. Before loading, the DNA is mixed with a loading dye that weighs down the sample in the solution, so it does not leave the well, and also includes a visible marker to track the progression of the run. Unknown samples are often run alongside a DNA Ladder, containing known lengths of DNA for comparison. The gel is then placed in a container, called a gel tank or box, filled with conductive, pH controlled buffer solution, usually Tris-acetate-EDTA or Tris-Borate-EDTA and an electrical field is applied along the length of the gel. The process is summarised in figure 2.

The speed of movement through the gel is then determined by the voltage gradient, i.e. the voltage between the electrodes. The required field strength is related to the size of the gel tank being used and the required voltage can be calculated using the simple equation E = V/d where E is the field strength, V the voltage and d the distance in cm between electrodes. Horizontal gel tanks are generally run at between 5 – 10 V / cm so if your tank has an electrode distance of 10 cm, you would run the gel at 50 – 100V. The exact value depends on your samples and should be determined empirically.

To apply this electrical field, we use a DC power supply. Most electrophoresis power supplies can be set to provide either a constant current or a constant voltage, with each having advantages and disadvantages. However, since agarose gel electrophoresis uses a continuous buffer system i.e. the anode, cathode and gel buffer are all the same, the variation of these parameters will yield the same results. We can simply set the power supply to constant voltage, based on the size of the tank as described above.

One potential issue is the production of heat due to the flow of current through the system which can be especially high with larger tanks that require higher voltage. For this reason, it is advisable to use some form of cooling, either passive in the form of a cooling block, or active such as a recirculating chiller, for larger electrophoresis systems.

Visualising the DNA

Figure 3: Image of an agarose gel stained with Ethidium Bromide and captured using a gel documentation system.

After the DNA has migrated through the gel, it needs to be visualised, so we can determine the length and abundance of the molecules in the sample. Since DNA is not visible to the naked eye, we stain it with either a coloured stain, such as methylene blue, or more frequently with a fluorescent stain such as Ethidium Bromide. Fluorescent stains give much better detection levels when imaging gels. There are a wide range of stains on the market, some being added to gel before casting and some being used to stain the gel after the run. Whichever stain you use, the next step is to capture an image in a gel documentation system.

Gel documentations systems, or gel docs for short, use high sensitivity cameras to capture images of the agarose gels. Often these systems are equipped with UV or blue light transilluminators, which are used to excite the fluorescent stains, which then emit light which can be captured by the camera. All gel docs will come with some form of illumination source, a filter to remove background light and a camera to detect the signal. Other than these basics there are a huge range of gel docs available starting from basic hood systems to systems with integrated PCs and touchscreens. Once you’ve captured an image, you can analyse it based on the pattern of the DNA bands to determine their length and the quantity of DNA present.

Video Demonstration

Applications

Given the simple nature of this technique, scientists have been able to apply it to a wide range of studies, some of which are discussed below.

Molecular Cloning

Probably the most frequent application of agarose gel electrophoresis is in molecular cloning. This is the construction of recombinant DNA molecules that are integrated into various organisms to create genetic modifications. The purpose of these modifications varies and can include production of a specific biomolecule, for example the production of insulin in pharmaceutical manufacturing. Other applications of molecular cloning include adding fluorescent protein fusions to existing cellular proteins to study their location in cells and creating new genetic circuits to carry out specific functions, such as breaking down toxins.

Whatever the desired end product is, electrophoresis is a key step in both the production and quality control of DNA fragments used in molecular cloning. Electrophoresis can be used to analyse the fragments created by polymerase chain reaction (PCR) or restriction digest, to ensure they are of the correct size. It can also be used to purify fragments, by running them on the gel and subsequently cutting out the band of interest and purifying the DNA from the agarose.

Genetic Fingerprinting

Combined with PCR, agarose gel electrophoresis can be a powerful technique for identifying individuals based on their genetic code. The human genome contains many regions of short repeats, the number of which vary uniquely between individuals. By targeting these regions with specific PCR primers, a profile of band on an electrophoresis gel corresponding to these regions can be created that is unique to that individual. This technique, known as DNA fingerprinting, can be used in areas such as forensics for criminal investigations, genealogy and parentage testing.

Diagnostics

Electrophoresis can be used in a range of diagnostic tests, primarily in the screening of genetic disorders but also to identify abnormal proteins. DNA can be extracted from patients, or even from embryos for pre-implantation screening, and subject to PCR and agarose gel electrophoresis to confirm the presence of certain genes or genetic abnormalities. Agarose gel electrophoresis can also be applied to some proteins, for example to study blood chemistry to determine suitability of certain medical treatments.

The wide range of applications, both academic and clinical make agarose gel electrophoresis an extremely important technique. Although the recent advent of next generation sequencing technologies has the potential to replace many of the current uses of agarose gels, their ease of use and versatility mean that this technique is likely to persist for the foreseeable future.

Equipment for Agarose Gel Electrophoresis

Figure 4: Diagram of a gel tank with all components.

The popularity of agarose gel electrophoresis is partly due to its simplicity. The equipment required is easy to use and takes little training to operate correctly. The main components are discussed below.

Gel Tank/Gel Box

The gel tank, also called a gel box, is the main component of the horizontal agarose gel electrophoresis system. Generally, a gel tank will consist of a plastic container with a raised centre platform where the gel is places on a secondary support called a gel tray. At either end of the tank, electrodes made from an inert conductive material, most commonly platinum, are fixed and wired to connectors to allow the connection to the power supply. Finally, a lid sits on the gel tank to prevent access to the chamber while high voltage is applied to the buffer.

Cleaver Scientific manufactures gel tanks in a range of sizes for different applications and can custom manufacture systems for niche applications. Take a look at the selection chart and browse our product pages for more information.

Power Supply

To apply an electrical field to the gel, you will need an electrophoresis power supply. These power supplies are specifically manufactured for electrophoresis applications and features very stable voltage and current outputs to prevent fluctuations in migrations speed. A good power supply with allow you to set either constant current or voltage depending on the requirement of the experiment, and more advanced supplies will allow programming of individual steps at different parameter values.

At Cleaver scientific we have a range of electrophoresis power supplies for all applications. The PowerPRO series of power supplies is a versatile range designed to power both multiSUB horizontal and omniPAGE vertical electrophoresis tanks. Each power supply has a 2.4″ LCD display. Constant voltage, current and power options are available as well as pre-programmed or customer programmed conditions allowing users to save and repeat their experiments for exceptional reproducibility.

Gel Documentation System

For the final stage of the technique, gel imaging, you will need a gel documentation system as described above. Cleaver Scientific have a whole range of gel documentations to suite any budget or requirement. Take a look at our selection guide to find the best option for you and browse our product pages for more information.

Reagents for Agarose Gel Electrophoresis

To run a gel electrophoresis experiment you will require both the equipment and the reagents. The basic reagents required for agarose gel electrophoresis are:

  • The agarose powder, to make the gel
  • Buffer stocks to make the running buffer
  • Loading dye to mix with DNA
  • DNA Ladders to compare DNA lengths
  • DNA stain for visualising DNA

Cleaver Scientific supplies all these reagents, include runSAFE, a non-toxic DNA stain that works with blue light for increased cloning efficiency and safety of use.