1. Classification of Mycobacteriophages With the Use of 2012
Cluster Specific Primers
González-Sánchez, Angélica M. , González-Sánchez, Pablo
RISE Program – University of Puerto Rico atCayey
Abstract
Bacteria, 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.
Introduction
Mycobacteriophages 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

2. 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 Methods
For 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.
Results
As 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
MycobacteriophageCemi was tested, the Mycobacteriophage appears to be part of cluster

3. 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
LorenzovegMycobacteriophage is present and expressed in the lane with the B2 cluster
specific primer. Therefore, the target DNA from MycobacteriophageLorenzoveg can be
tentatively assigned as belonging to the B2 cluster. (Great!Very detailed explanation of the
results.)
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 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.

4. 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
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
aboutMycobacteriophages, 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.
Acknowledgements
We 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.