Fat man in a footrace

Now that the massive string of PCRs and gels are finished, we're just starting on Denaturing Gradient Gel Electrophoresis. We used some of Bill's samples today, from a few other projects that he has been working on. The gels themselves are quite thin compared to the normal and vertical, rather than horizontal, and have their own difficulties, mainly in the loading process.

DGGE works on the basis of the actual base pair sequence in certain pieces of DNA being different from the others. For instance, in strands which contain lots of Guanine and Cytosine bonds, it will begin to denature, or 'melt' at a higher temperature than ones which contain more Adenine and Thymine. This is due to the fact that the G-C unit in DNA has 3 hydrogen bonds, while the A-T unit only has two. Since the DNA molecule overall has a negative charge, it will tend to be drawn down towards the bottom of the gel, due to electrical fields being applied.

The whole concept of gel electrophoresis is that these negatively charged DNA fragments, varying in length from anywhere between 250 to 1500 base pairs long, will move through the gel 'mesh' towards the positive electrode on one end of the apparatus. But just like a fat man in a foot race, the larger and larger pieces will fall behind progressively and form their own 'bands' in the gel, which is then visualized under UV light to confirm it's presence.

What DGGE does however, is that it has a chemical which, at just the right concentration, will cause these pieces of DNA to split apart, but only halfway. Imagine a very rudimentary ladder as a DNA analogue, but then cut it apart down the middle and splay the two ends out in a Y formation. Suddenly, the DNA fragment can no longer move through the gel, as it is now snagged on the molecular matrix. It's' sorta like walking through a crowd, but when someone gives you a signal you have to open your arms wide. Suddenly, you can no longer move nearly as quickly towards your goal.

Where they get stuck in the gel tells us a lot about it's overall sequence makeup, including how to differentiate between different mutations of the same region of DNA sequence. For instance, the technique is being used in cancer research to see whether or not certain mutated sequences of DNA are correlated with cancer. In the cell biology and microbiology realm, it has been used to differentiate between notoriously mutation-prone mycobaterium.

In our work, we're planning to use it to look for the number of different sequence mutations in our DNA samples, which gives us a big clue into how many different species of bacteria reside in the soil.