Post 4: tRFLP

Hello Everyone!

You may be wondering what the point is of all of the work in the previous posts. Where does this research all lead? What is she trying to determine? Well, it all leads right here, to Terminal Restriction Fragment Length Polymorphism or tRFLP.

In brief, tRFLP is a means of creating a community profile of the various assemblages of bacteria within Lake Matoaka, the Spillway, the Inlet and the soil around the lake. By digesting the terminally labelled PCR products of the 16s ribosomal rNA gene (a highly conserved gene present in all bacteria) with the same enzymes, the varying sizes of each fragment can be analyzed using capillary electrophoresis. Very simply, different fragment sizes indicate different groups of bacteria. Identical fragment sizes indicate identical or very similar groups of bacteria.

However, before capillary electrophoresis is carried out, the PCR amplicons must be purified, prepared and digested. Purification is done by means of a Qiagen kit (which I have heard referred to by one very wise professor as the “Miracle in the Blue Box”). The kit removes all of the Taq Polymerase, extra dNTPs and such from the amplicons.  These particles would inhibit the process of capillary electrophoresis. After PCR product purification, the amplicons are digested with Mung Bean nuclease. This is an enzyme that cleaves any single-stranded overhangs on the PCR products. Blunted, double-stranded DNA is ideal for a restriction digest. With the blunting complete, the DNA is digested with two enzymes HinfI and MspI. These two restriction enzymes recognize palindromic sequences of five and four base pairs respectively. Anywhere the enzyme recognizes this specific sequence of DNA is cleaves the DNA apart, creating smaller double-stranded fragments. These fragments are then used in capillary electrophoresis.

Capillary electrophoresis is carried out in the ABI3130 Genetic Analyzer. Essentially, the termini of these DNA fragments were labelled with the forward primer, 27f-HEX. This primer fluoresces green when hit by a laser. The capillary electrophoresis machine separates the fragments by size and funnels the fragments individually through the tubing. When each fragment passes the laser, the electrons in the fluorescent primer are excited and send a signal to the computer.  This signal creates an electropherogram that looks something like this:

Each peak represents a fragment size. The height of the peak indicates the number of fragments of that size. Any peaks that are the same for every sample indicate an overlap in the bacterial community.

Thus far, I have generated tRFLP electropherograms for the May, June and July soil and water samples. Detailed analysis has yet to be performed. However, preliminary analysis indicates that while there are bacteria groups present in all samples, there is a fair amount of divergence amongst the sample sites. It will be interesting to see who exactly is out there!

 Dana