Classification of Mycobacteriophages With the Use of Cluster Specific Primers 2012

González-Sánchez, Angélica M. , González-Sánchez, PabloRISE Program – University of Puerto Rico at Cayey

AbstractBacteria, the most numerous organisms on Earth, can be infected by viruses. Viruses

that infect bacteria of the mycobacteria genus are called Mycobacteriophages. Recent discoveries with these viruses have led to an understanding of their importance and how they are related to us. They relate to us because diseases like tuberculosis that affect the human population are caused by them . A good thing about working with these viruses is that they are easily found in bacteria that live in the soil. The purpose of this experiment was to isolate Mycobacteriophages found in Puerto Rican soil, identify their genomic sequence, and compare them to the genomic sequence of known viruses so that they could be classified under the same cluster or group. To accomplish this, Polymerase Chain Reaction (PCR) technique was used to amplify the DNA regions of the Mycobacteriophages. Then, gel electrophoresis was used to separate the molecules according to their mass and charge. An important factor in this technique was the cluster specific primers which would later help in the classification of the phage. The gels showed one certain result, many uncertain results, and some gels did not show any results at all. The certain result could contribute to other research being made about bacteriophages because now the phage is classified in a cluster and this means it shares the same properties as other bacteriophages in the same cluster.

IntroductionMycobacteriophages are viruses that infect bacteria from the mycobacteria genus.

One of the most common types of mycobacteria is the Mycobacterium smegmatis, which can be easily found in soil. By taking soil samples that contain the Mycobacterium smegmatis, Mycobacteriophages can also be found, isolated and studied. This can be very useful for a better understanding of evolution, genetics, structural biology and molecular interactions (Rubin, 2012, p.4). Dr. Michael Rubin, professor at the University of Puerto Rico in Cayey, has been researching Mycobacteriophages by isolating them from tropical soils of Puerto Rico, classifying them in clusters, and characterizing them with the use of bioinformatics tools. One of the most transcendental parts of this investigation is the sorting of Mycobacteriophages into clusters. Clusters are groups of Mycobacteriophages which have been taxonomically gathered due to their genome’s similarities. By identifying a Mycobacteriophage as belonging to a cluster, its genomic sequence can be more easily determined, annotated and analyzed. To sort a Mycobacteriophage into a cluster, molecular techniques are used. For example, as in the experiment described further on in this report, Polymerase Chain Reaction (PCR) is used for the amplification of genomic DNA by using cluster specific primers. These primers are precisely designed to bind only to complementary regions of DNA from a given Mycobacteriophage cluster. Therefore, by analyzing the products from the PCR, through an agarose gel electrophoresis, it will be possible to determine to which primer the studied target DNA binds, because of the expression of the bands in the gel. The expression of bands will mean that the

Mycobacteriophage’s DNA target region bound to the primer in the given electrophoresis gel lane. As a consequence, it is expected that Mycobacteriophage’s DNA will bind to a primer only if it is from the cluster for which the primer was designed. By identifying that type of complementarity, the Mycobacteriophage target DNA can be tentatively assigned to a cluster .

Materials and MethodsFor the PCR amplification of Mycobacteriophage genomic DNA regions for cluster

classification, first the Mycobacteriophage was prepared by isolating it. Then 1ml of the phage was transferred to a clean sterile microtube and centrifuged at 10,000 X g for 1hour at 4˚C. After this, 950 ul of supernatant were micropipetted to leave the concentrated Mycobacteriophage for use in the PCR. To prepare the primers, they were suspended in PCR Grade Water to a concentration of 10 ug/ul. After this was completed, the Polymerase Chain Reaction was set up by using the cluster specific primer combination, and adding it successively into a PCR reaction tube: 5 ul of nano pure PCR grade water, 5ul of the Mycobateriophage DNA from step #3, 1 ul of cluster specific forward primer, 1ul of reverse primer, and 12 ul of PCR master mix (Taq polymerase, buffer, nucleotides, Mg++). When the PCR reaction components were added, the PCR tubes were placed in the thermocycler for amplification. To amplify the DNA in the thermocycler, the mixture was left 5 minutes and 30 seconds at 95˚C for denaturation. Then, it was left 30 seconds at 62˚C for annealing. After that, it was left for 2 minutes at 72˚C for extension, and when this was completed, the steps were repeated for 25 cycles. Finally, the mixture was left for 7-10 minutes at 72˚C for final extension.

After the PCR technique was completed, a 2% agarose gel was prepared by the addition of 2 grams of agarose, 10 ml of 10x TAE gel running buffer and 90 ml of distilled water. Next, this mixture was microwaved on medium power, letting it boil for about 1 minute until the agarose was thoroughly dissolved. Using gloves, 4 ul of Ethidium Bromide were added to the mixture. Then it was briefly cooled and poured into a clean gel apparatus mold with the comb in place to form the wells. Once the gel was allowed to solidify for 30 minutes to 1 hour , 4 ul of loading dye were added to 34 ul of the PCR reaction . the next step wa to add 10 ul of the 1Kb marker and 10 ul of the 100bp marker in the first and last well of the 2% agarose gel respectively. Afterwards, 10 ul of each reaction mixture were loaded in each of the remaining wells. The gel was then put to run at 80 volts for 1 hour. After letting the gel run, it was photographed using a gel documentation system. Results were analyzed.

ResultsAs seen in figure 1, from the upper part of agarose gel 1, it can be determined that

the Mycobacteriophage called Bruce probably belongs to the B2 cluster because the gel showed a band in the lane that had the B2 cluster specific primer. Therefore, the cluster specific primer was complementary to regions of the phage’s DNA and that is why it amplified. However, these results are somewhat inconsistent because there was another lighter band expressed below the most noticeable one. Some other gels also showed uncertain results. For example, on the upper part of agarose gel 2, on which the Mycobacteriophage Cemi was tested, the Mycobacteriophage appears to be part of cluster B2, but since there is more than one band present, the result is not guaranteed. In the

lower part of this same gel, there is also some ambiguity about the results because there were bands showing DNA expression with more than one cluster specific primer. From the observations, one can assume that the Mycobacteriophage belongs to cluster E because it showed more expression in the lane with the cluster E specific primer. However, because it also showed expression with the B2 cluster specific primer, it cannot be classified as a part of any of these clusters. Another section that exhibited uncertainty was the lower part of agarose gel 3 because there were two bands present in the lane of the B2 cluster specific primer.

Aside from these uncertain results, there were some gels that showed no results at all such as the lower part of agarose gel 1 and both the upper and lower part of agarose gel 4.

The upper part of the agarose gel 3 is the one result from which it can be certainly determined to which cluster the Mycobacteriophage belongs. Here, the Lorenzoveg Mycobacteriophage is present and expressed in the lane with the B2 cluster specific primer. Therefore, the target DNA from Mycobacteriophage Lorenzoveg can be tentatively assigned as belonging to the B2 cluster. (Great! Very detailed explanation of the results.)

Discussion In this experiment, there were several different results. First of all, some of the gels

did not show any results at all in terms of bands. This could be caused by multiple factors such as infrastructural or equipment problems, unspecific primers, an incorrectly assembled reaction, non-functional reaction components, a target DNA that does not have complementary regions for the cluster specific primer used or because the Mycobacteriophage tested is from a cluster for which there are no specific primers. There were other gels that showed uncertain results whether because there was more than one band on a lane or in different lanes. This can probably be caused by experimental or human errors in the preparation of the reactions. Aside from this, there were also successful results in which a Mycobacteriophage could be assigned to a known cluster.

Also, because of technical problems with the thermocycler used for the PCR, there were some changes that had to be made in the experimental procedure and that occasioned

Figure 1: Results of the electrophoresis

analysis after PCR amplification of

Mycobacteriophage’s DNA with several different cluster specific primers.

alterations in the results. First of all, more reagents than specified by the original protocol had to be used. To make sure that the reaction would amplify correctly, additional 5 ul of water and 5 ul of master mix were added.

It can be concluded that by using cluster specific primers one can verify to which cluster the Mycobacteriophage belongs. This occurs because of the complementarity between the cluster specific primer and a DNA target region of a Mycobacteriophage from that cluster. It can also be determined that both experimental and technical factors can affect the results of the experiment.

Finally, this experiment can contribute significantly to current research about Mycobacteriophages, as for example the one being carried on by Dr. Michael Rubin. The accomplished classification of a Mycobacteriophage can be very useful in future experiments because it allows us to know about the genome and the characteristics of that Mycobacteriophage by the similarities shared with other Mycobacteriophages of that same cluster.

AcknowledgementsWe thank Dr. Michael Rubin for his invaluable instruction on the topic of

Mycobacteriophages and for providing the isolated Mycobacteriophages. We are also very thankful with Ms. Yadira Ortiz, our lab technician, who provided us all the materials needed for the experiment. We also thank the teaching assistants Ms. Valeria Rivera and Ms. Melisa Medina for all their technical support. Finally, we thank the RISE Program from the University of Puerto Rico in Cayey for giving us the opportunity to participate in this research.

Literature Cited Rubin, M. 2012. Cluster Classification of Mycobacteriophages Isolated From Tropical Soils of Puerto Rico. Cayey, P.R. University of Puerto Rico in Cayey.