1. Cluster Analysis and Classification of Mycobacteriophages
Wilmarie Morales Soto
Gretel S. Montañez Próspere
RISE Program Spring 2012
Abstract:
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 PCR
and Agarose Gel Electrophoresis techniques. Most phages that showed bands ended up belonging to
cluster 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 as
host bacterial species. Mycobacriophage structures consist of a head and tail. In its head the
mycobacteriophage stores it all its genetic material, which it injects into its host using its tail. Over
thousands of mycobacteriophages have been discovered to date, and they are classified using a cluster
system. They are organized into clusters that range from A through O (most containing sub clusters) using
their sequenced and annotated genomes as the bases for this classification.
For this workshop our mission was to take DNA from several micobacteriophages and classify
them into clusters using PCR and Gel Electrophoresis.
Materials and Methods:
In order to classify mycobacteriophages we implement two key methods which are polymerase
chain reaction (PCR) and Agarose Gel Electrophoresis. PCR is a replication technique that is used to
amplify specific segments of DNA. PCR consist of the denaturing, annealing, and extension of DNA

2. fragments at specific temperatures. For our PCR solution we transfer 5µl of a mycobacteriophage (in our
case Lorenoveg) into a clean microtube. We also added 5µl of pure PCR grade water. We added 1µl of
both, forward and reverse specific primers.For this experiment our specific primers belong to clusters A
through I. Finally we added our PCR master mix, which contains: Taq Polymerase (polymerase isolated
from species of bacteria founded in hot springs), buffer, nucleotides, Mg++. All of our PCR reactions were
placed in the thermo-cycler following the conditions of denaturing (95°C for 30 sec) annealing (62°C for
30 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 distilled
water. We added 4µl of loading dye to 10µl of our PCR reactions and we loaded them to our gels. We run
the 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, which
belonged to the primers for cluster B2. The gel for the phage Fenixious showed a band on the ninth lane

3. 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 gel
showed nothing. There are several reasons this might have happened. It could have been a result of
human error (bad pipetting, wrong primers, no master mix, etc.). Due to these reasons we could not use
this gel for our analysis.
The phages Bruce, Cemi, and Lorensoveg showed bands on the B2 primer lane. From this
knowledge we can conclude that all of this micophages belong to cluster B2. The mycophage Fenixious
showed 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 not
faulty. We can concludethat these micophages do not belong to clusters A through I. They may, on the
other hand, belong to a cluster from K to O, but since we did not test for these clusters we cannot safely
attest 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 gel
that 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 RISE
Program and its coordinators Dr. Robert Ross, Dr. Eneida Diaz and Dr. Elena Gonzalez
Literature Cited:

4. 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.