Cluster analysis and classification of mycobacteriophages
Cluster Analysis and Classification of Mycobacteriophages Wilmarie Morales Soto Gretel S. Montañez Próspere RISE Program Spring 2012Abstract: Mycobacteriaphages are members of a group of bacteriophages that use mycobacteria as hosts.Mycophages are organized into clusters in regards to their differences in sequence and annotated genomes.For this experiment we tried to classify several phages (Bruce, Carmin, Cemi, Fenixious, Lorensoveg,NovaAndreas, Phagius_Maximus, and Suave) in their respective clusters. To do this we used both PCRand Agarose Gel Electrophoresis techniques. Most phages that showed bands ended up belonging tocluster B2, and those that did not have bands most probably belong to clusters K through O.Introduction: Mycobacteriophages are members of a group of bacteriophages known to use mycobacteria ashost bacterial species. Mycobacriophage structures consist of a head and tail. In its head themycobacteriophage stores it all its genetic material, which it injects into its host using its tail. Overthousands of mycobacteriophages have been discovered to date, and they are classified using a clustersystem. They are organized into clusters that range from A through O (most containing sub clusters) usingtheir sequenced and annotated genomes as the bases for this classification. For this workshop our mission was to take DNA from several micobacteriophages and classifythem into clusters using PCR and Gel Electrophoresis.Materials and Methods: In order to classify mycobacteriophages we implement two key methods which are polymerasechain reaction (PCR) and Agarose Gel Electrophoresis. PCR is a replication technique that is used toamplify specific segments of DNA. PCR consist of the denaturing, annealing, and extension of DNA
fragments at specific temperatures. For our PCR solution we transfer 5µl of a mycobacteriophage (in ourcase Lorenoveg) into a clean microtube. We also added 5µl of pure PCR grade water. We added 1µl ofboth, forward and reverse specific primers.For this experiment our specific primers belong to clusters Athrough I. Finally we added our PCR master mix, which contains: Taq Polymerase (polymerase isolatedfrom species of bacteria founded in hot springs), buffer, nucleotides, Mg++. All of our PCR reactions wereplaced in the thermo-cycler following the conditions of denaturing (95°C for 30 sec) annealing (62°C for30 sec) and extension (72° C for 2 min) repeating for a total of 25 cycles. For the second part of our experiment we used Agarose Gel Electrophoresis. We used a 2%agarose gel that was made with 2 grams of agarose, 10ml of TAE gel running buffer, and 90ml of distilledwater. We added 4µl of loading dye to 10µl of our PCR reactions and we loaded them to our gels. We runthe gels at 80volts for 1 hour. Then we photograph the gels using a documentation system.Results: Gel #1 Gel #2 Gel #3 Gel #4 We had four agarose gels, as shown above, each containing two different phages to be examined.On the first gel we had Bruce (upper) and Carmina (lower). On the secondgel we had the phages Cemi(upper) and Fenixious (lower). Gels four and five contained phages Lorensoveg (gel 3- upper),NovaAndreas (gel- 3 lower), Phageus_Maximus (gel 4- upper), and Suave (gel 4-lower). The gels containing Bruce, Cemi, and Loresoveg all showed bands on the fourth lane, whichbelonged to the primers for cluster B2. The gel for the phage Fenixious showed a band on the ninth lane
that contained the primers for the E cluster. On the other hand the gels that held the phages Carmina,NovaAndreas, Phagius_Maximus, and Suave were unsuccessful in showing bands.Discussion: Overall our gels worked, with the exception of the gel for Carmina (gel number 1 lower). This gelshowed nothing. There are several reasons this might have happened. It could have been a result ofhuman error (bad pipetting, wrong primers, no master mix, etc.). Due to these reasons we could not usethis gel for our analysis. The phages Bruce, Cemi, and Lorensoveg showed bands on the B2 primer lane. From thisknowledge we can conclude that all of this micophages belong to cluster B2. The mycophage Fenixiousshowed a band for the E primers, meaning that it belonged to cluster E. Carmina, NovaAndreas, Phagius_Maximus, and Suave, were unsuccessful in showing any bands,but from the concentration gathered at the top of these gels we can determine that these solutions were notfaulty. We can concludethat these micophages do not belong to clusters A through I. They may, on theother hand, belong to a cluster from K to O, but since we did not test for these clusters we cannot safelyattest to this, and thus we discard these gels from our study. In order to determine to which cluster these micophages belong to. We have to run another gelthat contains the rest of the cluster primers to make a more accurate analysis.Acknowledgements:To Dr. Rubin and his assistances: Valeria and Melisa, to our laboratory assistant Yadira, and to RISEProgram and its coordinators Dr. Robert Ross, Dr. Eneida Diaz and Dr. Elena GonzalezLiterature Cited:
1. Hatfull G., Pedulla M., Jacobs-Sera D., et.al.,2006. Plos genetics. Exploring the Mycobacteriophage Metaproteome: Phages Genetics as an Educational Platform. Vol 2. 835-847.2. Rubin M.,2012.Cluster Classification of Mycobacteriophages Isolated from Tropical Soils of Puerto Rico.