Comparing Viral Communities Using RAPD-PCR and Gel Electrophoresis

These past few weeks I have been working on using the viral concentrates I made for RAPD-PCR and gel electrophoresis. To make a viral concentrate, I filter off the bacteria and centrifuge the water samples at 38,000 rpm to force the viruses to the bottom of the centrifuge tubes. I then re-suspend the viruses in a much smaller volume. I have 27 viral concentrates to make gels for, plus the water samples I will continue to collect until October.

I use RAPD-PCR because it allows me to look at viral communities (each band representing a species) even though their genomes may not be in genome banks. Because most virus genomes are not sequences, I cannot pick a primer to use for regular PCR. RAPD generates a unique profile or fingerprint for each sample. I can use ecology statistics programs to compare how similar or different each sample’s fingerprint is to any other fingerprint in my data set by looking for bands of the same length from sample to sample.

However, at the beginning of my endeavor, I could not get the gels to come out with useable data. Some of the lanes did not have bands and the ones that did had bands that were too close together, which can be seen below.

Gel Top 7:7:17

The solution was to stain the gel for twice as long, run the gel for an hour longer, and use a high resolution gel, which is more difficult to make, but gave me clear results. Below, the gels are have more clearly visible bands that are stained well and spread out enough. That being said, the first gel has some lanes that are very faint and those samples will have to go through RAPD-PCR and gel electrophoresis again.

Gel 7-18-17 Exposure 6.5 Gel 7-21-17 Exposure 3.5

I am now running gels with 9 viral concentrates from different samples at a time. Once I have run all of my samples, I can compare the bands by length to look at how related each viral community is. In my past research, I found that there was a relatedness of the viral communities temporally but not spatial. However, because I only sampled once a week for five weeks, I think there may be a more clear relationship that will be revealed by more long term sampling.

This time around, I have been collecting samples monthly since last October and will continue to this October for a comprehensive look at how viral communities change over the course of a year.