Analyzing Polymorphisms in Personal DNA at the Alu Intersection

in Chromosome 16 at PV92 locus

Cary Carrington

March 3, 2016
BIO360 Cellular and Molecular Biology
Dr. Gena Nichols
Introduction:

The human genome is an intricate and complicated chain of sequences with billions of
base pairs. Throughout the human genome, there are specific genes known as Alu
elements due to the presence of a recognition site for the restriction enzyme, Alu I. This
300 base pair section of DNA mostly exist in introns and can be found in various
locations and even in some cases in exons. These elements are still not fully understood,
but what is known is that they can replicate themselves through retrotransposon, an RNA
intermediate copied into a DNA segment and even though they have the ability to
replicate, scientists claim that only a few genes known as master genes can actually
replicate themselves.

In humans, Alu elements are fixed, where both chromosomes have the same insertion.
Although some can be considered dimorphic, which means it might not always be at the
same chromosomal location. In a group of individuals that would have these differences
in DNA sequences, it is known as polymorphisms. It is also possible for the Alu element
to be present on one or both of the homologous chromosomes, making someone
heterozygous or homozygous.

In this experiment, a dimorphic Alu insertion found on chromosome 16 at the PV92 locus
will be identified from personal cheek cells by the process of Polymerase Chain Reaction
(PCR). Three steps will be done to complete the PCR, denaturation, annealing, and
extension, in order to complete one full cycle of PCR. The product will be observed on
agarose gel to determine whether the DNA sample is homozygous, heterozygous, or null.
The hypothesis is that the personal DNA sample will be visible enough to determine
whether it is homozygous, heterozygous, or null.

Materials and Methods:

1.5 microcentrifuge tube PCR Tube

10mL Saline Solution PV92 Primer mix
Medicine cup Edvobead
Micropipette Electroporesis unit
Lysis buffer InstaStain Card
Centrifuge Latex gloves
Waterbath float Light Box
Supernatant

Begin by labeling the microcentrifuge tube with group number or initials. Obtain 10 mL
of saline solution and rinse mouth vigorously for 10 seconds; expel the solution back into
the cup. Transfer 1.5 mL of solution into the microcentrifuge tube. Centrifuge the
suspension for 2 minutes at full speed to pellet the cells. Pour off the supernatant and
repeat the centrifuge twice more. Using the micropipette and 140 lysis buffer, pipette
the cheek cells up and down to resuspend the cells. Place the cap back onto the tube and
place in the waterbath float to incubate at 55 for 15 minutes. Once complete, mix the
sample by flicking the tube vigorously. Incubate the sample for 15 minutes in a 99
waterbath. Centrifuge the cellular lysate for 2 minutes at a low speed, around 6000 rpm.
Transfer 80 of the new supernatant to a new microcentrifuge tube and place it in ice.

Label a PCR tube with the sample and initials. Add 20 PV92 primer mix, 5
extracted DNA, and the PCR EdvoBead to the PCR tube. Mix the PCR sample to
completely dissolve the bead. Centrifuge the sample for a few seconds for the sample to
collect at the bottom. Then, amplify the DNA through PCR cycling conditions with an ,
consisting of:
Initial denaturation at 94 for 5 minutes
94 for 30 seconds
65 for 30 seconds
72 for 60 seconds
Final Extension 72 for 4 minutes
The cycling conditions will be done a total of 32 cycles, then 5 of 10x gel loading
solution to the sample and place the tubes in ice

After making the agarose gel and allowing it to cool, place it into the electrophoresis gel
and load the entire sample into a well, making note of where the sample is. Place the
sample cover back on and connect the leads to the power source for the proper amount of
time. Once electrophoresis is complete, remove the gel and slide onto flat, protected
surface for staining. Add a few dabs of electrophoresis buffer to moisten the gel. With
gloves on, remove the protective sheet from the InstaStain and place over the sample.
With the gloved hand, remove air bubbles by firmly pressing fingers along the card. Let
the gel stain for 3-5 minutes, then remove the card and observe the gel over a light box.
Document observations found in the gel.

Results:

Figure 1: Group results and personal result

Well 1: EdvoQuick
DNA Ladder
Well 4: Personal
Sample
Discussion:

While comparing well 4, the personal sample, to well 1, the DNA ladder, there is only a
700 bp line present, meaning that the personal sample is homozygous. While comparing
the personal sample to other results, it appears that everyone had the same results and are
homozygous for the Alu insertion. In well 6 though, you cannot see a band, so the results
are inconclusive. Some errors that could have caused this to happen include PCR cycling
conditions not being done correctly, misuse of centrifuge, misuse of micropipette, and
perhaps even not swishing the saline solution enough in the beginning to get enough skin
cells. The hypothesis stated at the beginning was that the personal DNA sample will be
visible enough to determine whether it is homozygous, heterozygous, or null. From the
results of the experiment, it can be said that the hypothesis is proven true because the
personal sample was determined as homozygous.