Classification of Mycobacteriophages With the Use of 2012Cluster Specific PrimersGonzález-Sánchez, Angélica M. , González-Sánchez, PabloRISE Program – University of Puerto Rico atCayeyAbstract Bacteria, the most numerous organisms on Earth, can be infected by viruses. Virusesthat infect bacteria of the mycobacteria genus are called Mycobacteriophages. Recentdiscoveries with these viruses have led to an understanding of their importance and howthey are related to us. They relate to us because diseases like tuberculosis that affect thehuman population are caused by them . A good thing about working with these viruses isthat they are easily found in bacteria that live in the soil. The purpose of this experimentwas to isolate Mycobacteriophages found in Puerto Rican soil, identify their genomicsequence, and compare them to the genomic sequence of known viruses so that they couldbe classified under the same cluster or group. To accomplish this, Polymerase ChainReaction (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 andcharge. An important factor in this technique was the cluster specific primers which wouldlater help in the classification of the phage. The gels showed one certain result, manyuncertain results, and some gels did not show any results at all. The certain result couldcontribute to other research being made about bacteriophages because now the phage isclassified in a cluster and this means it shares the same properties as other bacteriophagesin the same cluster.Introduction Mycobacteriophages are viruses that infect bacteria from the mycobacteria genus.One of the most common types of mycobacteria is the Mycobacterium smegmatis, which canbe easily found in soil. By taking soil samples that contain the Mycobacteriumsmegmatis,Mycobacteriophages can also be found, isolated and studied. This can be veryuseful for a better understanding of evolution, genetics, structural biology and molecularinteractions (Rubin, 2012, p.4). Dr. Michael Rubin, professor at the University of PuertoRico in Cayey, has been researching Mycobacteriophages by isolating them from tropicalsoils of Puerto Rico, classifying them in clusters, and characterizing them with the use ofbioinformatics tools. One of the most transcendental parts of this investigation is thesorting of Mycobacteriophages into clusters. Clusters are groups of Mycobacteriophageswhich have been taxonomically gathered due to their genome’s similarities. By identifyinga Mycobacteriophage as belonging to a cluster, its genomic sequence can be more easilydetermined, annotated and analyzed. To sort a Mycobacteriophage into a cluster, moleculartechniques 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 usingcluster specific primers. These primers are precisely designed to bind only tocomplementary regions of DNA from a given Mycobacteriophage cluster. Therefore, byanalyzing the products from the PCR, through an agarose gel electrophoresis, it will bepossible 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 theMycobacteriophage’s DNA target region bound to the primer in the given electrophoresisgel lane. As a consequence, it is expected that Mycobacteriophage’s DNA will bind to aprimer only if it is from the cluster for which the primer was designed. By identifying thattype of complementarity, the Mycobacteriophage target DNA can be tentatively assigned toa cluster .Materials and Methods For the PCR amplification of Mycobacteriophage genomic DNA regions for clusterclassification, first the Mycobacteriophage was prepared by isolating it. Then 1ml of thephage was transferred to a clean sterile microtube and centrifuged at 10,000 X g for 1hourat 4˚C. After this, 950 ul of supernatant were micropipetted to leave the concentratedMycobacteriophage for use in the PCR. To prepare the primers, they were suspended inPCR Grade Water to a concentration of 10 ug/ul. After this was completed, the PolymeraseChain Reaction was set up by using the cluster specific primer combination, and adding itsuccessively into a PCR reaction tube: 5 ul of nano pure PCR grade water, 5ul of theMycobateriophage DNA from step #3, 1 ul of cluster specific forward primer, 1ul of reverseprimer, and 12 ul of PCR master mix (Taq polymerase, buffer, nucleotides, Mg ++). When thePCR reaction components were added, the PCR tubes were placed in the thermocycler foramplification. To amplify the DNA in the thermocycler, the mixture was left 5 minutes and30 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, thesteps were repeated for 25 cycles. Finally, the mixture was left for 7-10 minutes at 72˚C forfinal extension. After the PCR technique was completed, a 2% agarose gel was prepared by theaddition of 2 grams of agarose, 10 ml of 10x TAE gel running buffer and 90 ml of distilledwater. Next, this mixture was microwaved on medium power, letting it boil for about 1minute until the agarose was thoroughly dissolved. Using gloves, 4 ul of Ethidium Bromidewere added to the mixture. Then it was briefly cooled and poured into a clean gel apparatusmold with the comb in place to form the wells. Once the gel was allowed to solidify for 30minutes to 1 hour , 4 ul of loading dye were added to 34 ul of the PCR reaction . the nextstep wa to add 10 ul of the 1Kb marker and 10 ul of the 100bp marker in the first and lastwell of the 2% agarose gel respectively. Afterwards, 10 ul of each reaction mixture wereloaded 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. Resultswere analyzed.Results As seen in figure 1, from the upper part of agarose gel 1, it can be determined thatthe Mycobacteriophage called Bruce probably belongs to the B2 cluster because the gelshowed a band in the lane that had the B2 cluster specific primer. Therefore, the clusterspecific primer was complementary to regions of the phage’s DNA and that is why itamplified. However, these results are somewhat inconsistent because there was anotherlighter band expressed below the most noticeable one. Some other gels also showeduncertain results. For example, on the upper part of agarose gel 2, on which theMycobacteriophageCemi 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 thelower part of this same gel, there is also some ambiguity about the results because therewere bands showing DNA expression with more than one cluster specific primer. From theobservations, one can assume that the Mycobacteriophage belongs to cluster E because itshowed more expression in the lane with the cluster E specific primer. However, because italso showed expression with the B2 cluster specific primer, it cannot be classified as a partof any of these clusters. Another section that exhibited uncertainty was the lower part ofagarose gel 3 because there were two bands present in the lane of the B2 cluster specificprimer. Aside from these uncertain results, there were some gels that showed no results atall such as the lower part of agarose gel 1 and both the upper and lower part of agarose gel4. The upper part of the agarose gel 3 is the one result from which it can be certainlydetermined to which cluster the Mycobacteriophage belongs. Here, theLorenzovegMycobacteriophage is present and expressed in the lane with the B2 clusterspecific primer. Therefore, the target DNA from MycobacteriophageLorenzoveg can betentatively assigned as belonging to the B2 cluster. (Great!Very detailed explanation of theresults.) Figure 1: Results of the electrophoresis analysis after PCR amplification of Mycobacteriophage’s DNA with several different cluster specific primers.Discussion In this experiment, there were several different results. First of all, some of the gelsdid not show any results at all in terms of bands. This could be caused by multiple factorssuch as infrastructural or equipment problems, unspecific primers, an incorrectlyassembled reaction, non-functional reaction components, a target DNA that does not havecomplementary regions for the cluster specific primer used or because theMycobacteriophage tested is from a cluster for which there are no specific primers. Therewere other gels that showed uncertain results whether because there was more than oneband on a lane or in different lanes. This can probably be caused by experimental or humanerrors in the preparation of the reactions. Aside from this, there were also successfulresults in which a Mycobacteriophage could be assigned to a known cluster.
Also, because of technical problems with the thermocycler used for the PCR, therewere some changes that had to be made in the experimental procedure and that occasionedalterations in the results. First of all, more reagents than specified by the original protocolhad to be used. To make sure that the reaction would amplify correctly, additional 5 ul ofwater and 5 ul of master mix were added. It can be concluded that by using cluster specific primers one can verify to whichcluster the Mycobacteriophage belongs. This occurs because of the complementaritybetween the cluster specific primer and a DNA target region of a Mycobacteriophage fromthat cluster. It can also be determined that both experimental and technical factors canaffect the results of the experiment. Finally, this experiment can contribute significantly to current researchaboutMycobacteriophages, as for example the one being carried on by Dr. Michael Rubin.The accomplished classification of a Mycobacteriophage can be very useful in futureexperiments because it allows us to know about the genome and the characteristics of thatMycobacteriophage by the similarities shared with other Mycobacteriophages of that samecluster.Acknowledgements We thank Dr. Michael Rubin for his invaluable instruction on the topic ofMycobacteriophages and for providing the isolated Mycobacteriophages. We are also verythankful with Ms. Yadira Ortiz, our lab technician, who provided us all the materials neededfor the experiment. We also thank the teaching assistants Ms. Valeria Rivera and Ms. MelisaMedina for all their technical support. Finally, we thank the RISE Program from theUniversity of Puerto Rico in Cayey for giving us the opportunity to participate in thisresearch.Literature CitedRubin, M. 2012. Cluster Classification of Mycobacteriophages Isolated From Tropical Soils ofPuerto Rico. Cayey, P.R. University of Puerto Rico in Cayey.