Cluster Classification of Mycobacteriophages Isolated From Tropical Soils of Puerto Rico

Carolina Montañez and Natalia Manzano

RISE Laboratory in the University of Puerto Rico at Cayey

Abstract Some mycobacteriophages –viruses of the mycobacteria– isolated from the soils of Puerto Rico were classified

into clusters using Polymerase Chain Reaction, for amplification of the genome, and Gel Electrophoresis for

observing and comparing the results. For this classification there were 13 primers used, which must be

complementary to spaced regions that will be attached to the area that will be amplified. Also there was a

control used, Lilac, which is a member of Cluster E. After observing and comparing the migration patterns of

the results, 4 mycobacteriohages were classified, three in cluster B2 and one in cluster E. Bruce, Cemi and

Lorenzoveg are now part of Cluster B2 and Fenixious is a member of Cluster E.

Introduction Mycobacteriophages, viruses of the mycobacteria, have a special structure composed of a head, which

contains DNA, and the tail which is used to inject the genetic material into the host. But phages can readily

switch or expand their host range (Graham et. al. 2006). These mycobacteriophages are highly diverse and are

a very abundant life form. These are characterized based in their morphology and genome; using proteomic

techniques they can be classified in clusters using genomic sequence comparisons. First, they have to be

isolated from soil, identified and then plaque purified.

To be classified in clusters they have to share similar characteristics based on their sequence and annotated

genomes (Rubin 2012). In order to classify them first it must be used some techniques like Polymerase Chain

Reaction and Gel Electrophoresis.

Polymerase Chain Reaction is used to amplify and create copies of small quantities of a specific segment of

DNA (Medicinet 2012). That is why it’s used specific DNA primers, which must be complementary to spaced

regions that attached to the area that will be amplified. Using Gel Electrophoresis, which is a technique for

molecular separation based on charge and mass using an electric field, it can be determined if the

Mycobacteriophage belongs to the a cluster (Absolute Astronomy 2011). This can be known if it is present

similar migration patterns that allow cluster assignment and phage classification.

In the University of Puerto Rico at Cayey for many years they have been isolating, purifying and classifying

mycobacteriophages found in different tropical soils. Our aim is to classify the mycobacteriophage Fenixious,

isolated from a tropical soil in Puerto Rico, in a specific cluster using molecular techniques. Using the correct

techniques we are convinced that the mycobacteriophage, Fenixious, will be classified in a cluster or sub

cluster.

Material and Methods

This experiment was based on cluster classification using the Polymerase Chain Reaction (PCR) technique and

Gel Electrophoresis. In the procedure, we first transferred mycobacteriophage HTPL to a microtube, and then

it was centrifuged for the concentration of mycobacteriophage particles. The use of a micropipette was

necessary for the aspiration of a supernatant, and later the primers were prepared. To set up the PCR it was

necessary the making of reagents that contained: water, the mycobacteriophage Fenixious, forward and

reverse cluster primers, and the PCR Master Mix (Taq Polymerase, Buffer, Nucleotides, Mg) for a total volume

of 34 μl. One of the reagents had a problem because of errors in dispersing solution, the 14th tube contained

cluster primers A1 and A2. Also the control reagent was made with the micobacteriophage Lilac, which is a

Cluster E member. After making the 16 reagents, these tubes were placed in the thermo cycler for the PCR to

begin the denaturation, annealing and amplification process. For gel electrophoresis it was used a 2% agarose

gel that was runned for an hour at 80 volts. Then results were analyzed and a picture of the gel was taken.

Results and Discussion

After carefully and patiently working with Mycobacteriophages, by observing the agarose gel, we discovered

that some of them could be classified in different clusters. Bruce, Cemi and Lorenzoveg (Mycobacteriophages

studied by other class members) were classified in Cluster B2 and Fenixious in Cluster E. Surprisingly the

control mycobacteriophage Lilac is a member of Cluster E, just like Fenixious. This was determined after

observing the gel that contained 15 wells, one or two markers and 13 cluster forward and reverse primers.

These were classified in those clusters because of the special characteristics they shared on the genome. And

although tube 14th had A1 and A2 primers, this didn’t make any drastic changes on the migration pattern, it

simply did not showed up on the gel just like the rest of the primers that Fenixious was not part of them.

Fig. 1 This figure presents our agarose gel showing the migration pattern of Fenixious, which is now classified as part of Cluster E.

Acknowledgments

We want to thank Dr. Michael Rubin and his students Valeria Rivera and Melisa Medina for all their support

and help during this workshop. Also we will like to thank Yadira Santiago for preparing all the materials

needed, specially the gels that are very difficult to make. Finally we want to thank The RISE Program, Prof.

Eneida Díaz and Prof. Elena Gonzalez, for guiding us through this journey and helping us achieve our goals.

Bibliography

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