TECHNICAL ARTICLES

Download a PDF of this article

Principles and Applications of DGGE

Denaturing Gradient Gel Electrophoresis (DGGE) is a tool that was developed to analyze DNA. DNA is a molecule that is a double helix consisting of two, complimentary strands that resemble a clothing zipper that has been twisted along its axis. DGGE is used to detect changes (mutations) in the genetic code within a sample, and can detect as little as one base pair difference between strands of DNA.

How the Process Works – Mutations
The principle of this technique is to separate DNA strands, based on their actual base composition, or the ratio of GC to AT base pairs that make up a particular segment of DNA. This is accomplished by exposing the DNA to a gradient of denaturant at elevated temperatures within a polyacrylamide gel.

As the DNA sample progresses through the gel, from a low denaturant concentration to a higher one, it starts to melt at varying points. This is akin to the DNA “unzipping.” The higher the GC content of the sample, the harder it is to melt. Thus, the DNA sample is able to progress further into the gel before stopping. Samples with lower GC content melt more rapidly in comparison. Therefore, they progress more slowly within the gel, thus becoming separated from the other faster moving strands of DNA.

Other Applications – Source Tracking Contamination
In addition to mutational analysis, DGGE is a useful tool for source tracking contamination. For example, if a certain microbial contaminant is appearing in a particular product, DGGE can be used to track this organism to its source. This can be particularly useful in production facilities for the food, pharmaceutical, and various manufacturing systems industries.

Segments of DNA for all species of bacteria or fungi in the source sample are amplified by PCR (polymerase chain reaction). Typically, amplification is performed with genes or segments of DNA that are common to all the organisms that may be present. Even though these genes are highly conserved, there exists enough variation within regions of these genes and between species that they can be used to identify the organism from which they originated. Using DGGE, the amplified segments of DNA can be separated into the individual species bands, recovered, and then used for sequencing to identify each organism within the sample.

The banding pattern itself can be compared back to the original source sample for each analyzed sample in order to visualize which areas along a production line are showing either the same pattern, or that have the same contaminating band as the original sample. This is a rapid and economical way of comparing large numbers of samples to one another without having to culture, isolate, and analyze each sample individually.

The ability of DGGE to separate individual species within a sample also enables one to follow the progression of communities over a period of time. This application is extremely useful for remediation studies. In those cases, sample sites require sampling over extended periods of time to follow the degradation of contaminants and the organisms degrading them.

The primary benefit of DGGE is that the progression of specific organisms within the community or the overall community itself can be followed and catalogued rapidly without the use of expensive media or reagents. The greatest problem for traditional studies is that most of the organisms do not have the same growth requirements, thus requiring large sample volumes, multiple medias, and several weeks to get the organisms to grow (if they can even be cultured at all). DGGE eliminates several of these problems by eliminating the need for media, minimizing the sample volumes, and being able to detect organisms that are either unculturable or that may have perished during transit from the sample site to the lab. DGGE also allows the researcher to examine which organisms are forced out of a community over time, which organisms are stable, and what new organisms may be appearing.

What DGGE Means to You
The capabilities of DGGE analysis, compared to traditional microbiological methods for source tracking or profiling, are clear. DGGE eliminates the need for expensive medias and thereby allows more samples to be processed for less money. It allows the researcher to work with a stable, and in most cases less pathogenic, form of the organism. DGGE cuts overall project costs, as well as the required time for the analysis (in many cases from a few weeks to just a few days). The ability of DGGE to save not only time and money, but also human resources, is probably one of the greatest benefits of this technology. It permits a company to do more with less, and gain a greater knowledge and understanding of the problems they may face.

Download a PDF of this article

For more information, contact:
Dr. Kim Baughman, Director of Development
Microbac Laboratories, Inc.
kbaughman@microbac.com

© 2005-2010 Microbac Laboratories, Inc. All rights reserved. Webdesign Team