Agarose gel electrophoresis

Encyclopedia : A : AG : AGA : Agarose gel electrophoresis

Digital printout of an agarose gel electrophoresis of cat-insert plasmid DNA

Agarose gel electrophoresis is a method used in molecular biology to separate DNA strands by size, and to estimate the size of the separated strands by comparison to known fragments (DNA ladder). This is achieved by pulling negatively charged DNA molecules through an agarose matrix with an electric field. Shorter molecules move faster than longer ones.

Contents

1 Factors affecting migration
2 Visualisation: EtBr & dyes
3 Resolution limits
4 Buffers
5 Material
6 Preparation
7 Procedure
8 Analysis
9 References
10 See also

Factors affecting migration

The most important factor is the length of the DNA molecule with shorter double-helices traveling faster. But conformation of the DNA molecule is also important. For example, forms of a plasmid move with different speeds (slowest to fastest): open circular, linearised, supercoiled plasmid. Increasing the agarose concentration of a gel reduces the migration speed and enables separation of smaller DNA molecules. The presence of ethidium bromide (EtBr) in the gel causes DNA to run slower, as EtBr intercalates and uncoils DNA. The higher the voltage, the faster DNA migrates. But voltage is limited by the fact that it heats and ultimately causes the gel to melt. High voltages also decrease the resolution (above about 5 to 8 V/cm).

Visualisation: EtBr & dyes

The central dye in agarose gel electrophoresis is ethidium bromide, usually abbreviated as EtBr. It has the unique property of fluorescing under UV light when intercalated with DNA. By running DNA through an EtBr-treated gel and exposing it to UV light, distinct bands of DNA become visible.

Loading buffers are added with the DNA in order to visualize it and sediment it in the gel well. Negatively charged indicators keep track of the position of the DNA. Xylene cyanol and Bromophenol blue are typically used. They run at about 5000 bp and 300 bp respectively, but the precise position varies with percentage of the gel. Other less frequently used progress markers are Cresol Red and Orange G which run at about 125 bp and 50 bp.

Resolution limits

DNA-based gel electrophoresis can be used for the separation of DNA fragments of 50 base pairs up to several megabases (millions of bases). Large DNA molecules are only able to move end on in a process called "reptation" and are more difficult to separate. In general the lower the concentration of agarose, the larger is the ideal size of a molecule to be resolved up to 750,000 bp. The disadvantage of lower concentrations is the long run times (sometimes days) and the problem of handling the fragile gel.

Buffers

There are a number of buffers used for agarose electrophoresis. The most common being: tris acetate EDTA (TAE), Tris/Borate/EDTA (TBE)Sambrook J, Russel DW (2001). Molecular Cloning: A Laboratory Manual 3rd Ed. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY. and Sodium boric acid (SB). TAE has the lowest buffering capacity but provides the best resolution for larger DNA. This means a lower voltage and more time, but a better product. SB is relatively new and is ineffective in resolving fragments larger than 5kb; However, with its low conductivity, a much higher voltage could be used (up to 35 V/cm), which means a shorter analysis time for routine electrophoresis. As low as one base pair size difference could be resolved in 3% agarose gel with an extremely low conductivity medium (1 mM Lithium borate)Brody JR, Calhoun ES, Gallmeier E, Creavalle TD, Kern SE (2004). Ultra-fast high-resolution agarose electrophoresis of DNA and RNA using low-molarity conductive media. Biotechniques. 37:598-602. [link].

Material

For an agarose gel electrophoresis, several items are needed:

The DNA that is to be separated.
A DNA ladder, a mixture of DNA fragments (usually 10-20) of known size. The size of the DNA strands that are separated is determined by comparison of their relative position to that of the DNA strands of the DNA ladder. There are several DNA ladder mixes commercially available.
Buffer solution, usually TBE or TAE 1.0x, pH 8.0
Agarose
Ethidium bromide (5.25 mg/ml in H₂O)
Nitrile gloves
A color marker containing a low molecular weight dye such as "bromophenol blue" (to enable tracking the progress of the electrophoresis) and glycerol (to make the DNA solution more dense so it will sink into the wells of the gel).
A gel rack
A "comb" (usually cut from a sheet of teflon)

Preparation

There are several methods for preparing agarose gels. A common example is shown here. Other methods might differ in the buffering system used, the sample size to be loaded, the total volume of the gel (typically thickness is kept to a minimum while length and breadth are varied as needed), and whether the gel is prepared horizontally or vertically (the vast majority of agarose gels used in modern molecular biology are prepared and run horizontally).

Make a 1% agarose solution in 0.5x TBE. If you analyze small DNA strands, go up to 2%. Use 15-70 ml, depending on the size of the gel.
Boil solution, preferably in a microwave oven.
Let the solution cool down to about 60 °C at room temperature. Stir the solution while cooling.
Add 1 ul ethidium bromide per 10 ml gel solution. Wear gloves from here on, ethidium bromide is a potent mutagen (nitrile gloves recommended) ! Some researchers prefer not to add ethidium bromide to the gel itself, instead soaking the gel in an ethidium bromide solution after running.
Stir the solution to disperse the ethidium bromide, then fill it into the gel rack.
Insert the comb at one side of the gel, about 5-10 mm from the border of the gel.
When the gel has cooled down and become solid, remove the comb. The holes that remain in the gel are the slots.
Put the gel, together with the rack, into a chamber with 0.5x TBE. Make sure the gel is completely covered with TBE, and that the slots are at the electrode that will have the negative current.
Add the color marker to the DNA ladder is usually already stained.

for more information on ethidium bromide safety see references States, Kelly M. (2003). Ethidium Bromide in [The Waste-Paper:The Hazardous Waste Disposal Monthly Update]. Retrieved 2005-01-31.,Office of Biological Safety, Univ. of Wisconsin (Madison) (2003). Ethidium Bromide: Alternatives and Safe Handling in [BioSide Lines:The Newsletter of the UW Office of Biological Safety]. Retrieved 2005-01-31.,Environmental Health and Safety at The Scripps Research Intititute (1999). WILL YOUR GLOVES PROTECT YOU? in [Environmental Health & Safety:Second Quarter 1999] Retrieved 2005-01-31.

for information on alternatives to ethidium bromide see references ,Madden, Dean (2004 [last modified]). [Safer stains for DNA]. Retrieved 2005-01-31.

Procedure

After the gel has been prepared, use a micropipette to inject about 25 µl of stained DNA (a DNA ladder is also highly recommended). Close the lid of the electrophoresis chamber and apply current (typically 100 V for 30 minutes with 15 ml of gel). The colored dye in the DNA ladder and DNA samples acts as a "front wave" that runs faster than the DNA itself. When the "front wave" approaches the end of the gel, the current is stopped. It is now possible to visualize the DNA (stained with ethidium bromide) with ultraviolet light.

Figure 1: Schematic drawing of the electrophoresis process, see text for description of steps

Steps:

The agarose gel with three slots (S).
Injection of DNA ladder (molecular weight markers) into the first slot.
DNA ladder injected. Injection of samples into the second and third slot.
A current is applied. The DNA moves toward the positive anode due to the negative charges on its phosphate backbone.
Small DNA strands move fast, large DNA strands move slowly through the gel. The DNA is not normally visible during this process, so the marker dye is added to the DNA to avoid the DNA being run entirely off the gel. The marker dye has a low molecular weight, and migrates faster than the DNA, so as long as the marker has not run past the end of the gel, the DNA will still be in the gel.
The DNA is spread over the whole gel. The electrophoresis process is finished.

Analysis

Modern day gel electrophoresis research often leverages software-based image analysis tools, such as those used in two-dimensional gel electrophoresis, or 2-DE. In proteomics research, these tools primarily analyze bio-markers by quantifying individual, and showing the separation between one or more protein "spots" on a scanned image of a 2-DE product. Additionally, these tools match spots between gels of similar samples to show, for example, proteomic differences between early and advanced stages of an illness. Two leading tools in this study are [PDQuest] and [Progenesis Workstation]. While this technology is widely utilized, the intelligence has not been perfected yet. For example, while both of the aforementioned tools tend to agree on the quantification and analysis of well-defined well-separated protein spots, they deliver different results and tendencies with less-defined less-separated spots.Arora, Pankaj S., et al. (2005). Comparative evaluation of two two-dimensional gel electrophoresis image analysis software applications using synovial fluids from patients with joint disease. Journal of Orthopaedic Science 10(2):160-166. [link]

Illuminate the gel with an ultraviolet lamp (usually by placing it on a light box) to view the DNA bands - ethidium bromide fluoresces pink in the presence of DNA. Use a suitable UV shield or wear a UV protective face shield and cover all exposed skin (Standard laboratory gloves and glasses are not sufficient-don't forget to cover your neck!). The DNA band can also be cut out of the gel, and can then be dissolved to retrieve the purified DNA.