Experiment 2D is mainly about the Polymerase Chain Reaction or PCR.

PCR

In the experiment, the collected cheek samples were added with InstaGene™ matrix prior to
lysis. This matrix contains Chelex, a negatively charged microscopic bead which binds or
chelates to cellular metal ions (such as Mg+2) that are known as cofactors of enzymatic
reactions. When the cells are lysed in the presence of the chelating beads, the cofactors are
adsorbed and are not available to DNA degradation enzymes. The incubation of the mixture at
56OC for 10 minutes softened the plasma membrane, released clumps of cells from each other
and inactivated enzymes (e.g., DNAses) that tend to degrade DNA. Meanwhile, the incubation
at 100OC for 5 minutes denatured proteins and ruptured the remaining cell membranes resulting
to the release of DNA. The mixture was then centrifuged and the supernatant was collected.
The pellet was discarded since this contains the matrix bead that could inhibit the PCR reaction.

The complete master mix was then prepared and was added to the PCR mixture. This was
composed of the PCR master mix and the PCR primer solution. The master mix contains the
reagents needed for PCR amplification: (1) deoxyribonucleotide triphosphates, (2) recombinant
Taq DNA polymerase and (3) Mg2+ source. The Taq DNA polymerase, an enzyme that could
withstand high temperature, was used to synthesize new strands of DNA. The master mix also
contains red and yellow loading dyes which are responsible for the master mix’s color and
glycerol which made the samples sink. The PCR primer solution was used for it allowed binding
to one specific site of the human DNA.

Primers are short segments of oligonucleotides used in many biochemical and molecular
techniques such as PCR and DNA sequencing. The sequence of the primer is complementary
to the template strand or target DNA sequence. These primers serve as “starters” for the DNA
polymerase to extend the target sequence. In PCR, forward and reverse primers are used to
further increase amplicon yield. The forward primer (5’ – 3’) attaches and initiates the synthesis
in the template strand (3’ – 5’), while the reverse primer (3’ – 5’) allows the DNA sequence
extension in the coding strand (5’ – 3’). Designing primers for the PCR is a necessary and
critical step for a successful amplification of the target DNA. Certain characteristics of primers
are essential to promote proper annealing as well as amplification. The primers should be 17 to
28 nucleotide bases in length to allow a more specific binding sequence. Also, the sequence
has to contain a high GC content of around 40 – 60%. The relatively high GC composition
allows that the melting temperature is above 50°C. In addition, the GC pairs significantly affect
the melting temperature by the equation, Tm (in °C) = 2(A+T) + 4(G+C). This equation called
the Wallace rule, estimates the melting temperature of an oligonucleotide. The melting points of
the primer must be in the range of around 52 - 62°C for double helical stability, and that at
higher temperatures, the primers tend to form secondary annealing structures and cannot bind
with the complementary strand. Aside from these, the primers added should not be
complementary to one another since the primer – primer interaction would compete with the
target sequence and may result in DNA amplification failure wherein the primer was sequenced
rather than the target. Finally, the primer should not be complementary to itself to prevent and
eliminate secondary structures. Having complementary sequences in the primer would tend for
the primer to bind with itself and prevent the interaction with the target sequence, which leads to
DNA amplification failure. Careful primer sequences are crucial in the success of PCR.

Forty cycles of amplification were done. Each cycle was as follows: 94ᴼC for 1 minute, 60ᴼC for
1 minute and 72ᴼC for 1 minute. These temperatures were all crucial: 94ᴼC was when the DNA
denatured into single strands, 60ᴼC was when the primers annealed on either side of the target
region to their complementary sequences and 72ᴼC was when Taq polymerase bound and
made a complimentary DNA strand to the single stranded DNA.
The amplified PCR samples were then subjected to agarose gel electrophoresis. But prior to
this, PV92 X loading dye were added. The agarose gel was then stained with Fast Blast stain.

The region of the DNA that was used for this experiment was from the PV92 locus of
chromosome 16. This part has a short single dimorphic Alu intron which means that this
element may be present in some but not in others. Some people may be homozygous (+/+)
(they have the insert in both homologous chromosomes), hmomozygous (-/-) (they do not have
the insert in either chromosome) or heterozygous (+/-) (they have the insert in one of their
chromosome). The presence or absence of this insert was detected using the polymerase chain
reaction followed by agarose gel electrophoresis.

The primers used flank both the entire Alu insertion (300 base pairs in length) and 641 base
pairs of the PV92 locus to amplify a 941 base pair fragment (if the Alu element is present) or a
641 base pair fragment (if the Alu element is absent). Agarose gel electrophoresis of the PCR
products is sufficient to distinguish among homozygotes (+/+) for the presence of the Alu repeat
(941 base pair product only), homozygotes (–/–) for the absence of the Alu repeat (641 base
pair product only), and heterozygotes (+/–) having both the 641 and the 941 base pair products.

Figure . Genotype of PV92 locus of chromosome 16.

Image source: from http://www.hanoverhigh.us/ departments
/science/rm215/genetics/pdf/labs/pcr_alu.pdf

However, the gel for the experiment did not turn out correctly; hence, the theoretical gel was
used for the discussion.

Figure . Theoretical Data and Gel.

Image source: from http://www.hanoverhigh.us/ departments
/science/rm215/genetics/pdf/labs/pcr_alu.pdf

The heterozygous sample the smaller 641 base pair band is more intense than the larger 941
bp band. This difference is due to the fact that the smaller fragment is amplified more efficiently
than the larger fragment. Copies of the shorter fragment can be made at a faster rate than the
bigger fragment, so more copies of the shorter fragment are created per cycle.

In the theoretical gel, students 1, 3 and 4 had one visible band while student 2 had two bands.
For student 2, the appearance of two bands meant that he/she is heterozygous (+/-) for the
PV92 Alu insertion. Two students showed homozygous (+/+) for PV92 Alu insertion; they were
students 1 and 3. Meanwhile, student 4 was the only one who was homozygous (-/-) for the
absence of the PV92 Alu insertion.

The value of this Alu insert residing in a noncoding region of PV92 is that it does not code for
any protein sequence and is not related to a particular disease. Because Alu repeats have
inserted randomly in humans, the Alu insert in the PV92 locus can be very useful in studies of
allele and genotype frequencies in the human population. The principles of the Hardy-Weinberg
theory can be applied to analyze genotypic and allelic frequencies of the Alu insert in
populations. By determining the genotypic frequencies of the Alu genotype within your student
population, the corresponding allelic frequencies can also be calculated. Additionally, the
genotypic frequencies of your class population can be compared to published results of larger
population sizes.

Because Alu repeats appear in the general population at random, the Alu insert in chromosome
16 is very useful for the study of gene frequencies in localized human populations. Theoretically,
in some small, geographically isolated populations, all individuals may be homozygous +/+. In
others, the individuals may all be homozygous –/–. In a “melting-pot” population, the three
genotypes (+/+, +/–, –/–) may exist in equilibrium.

Reference:
Chromosome 8: PCR Wprkshop. Retrieved from http://pchs.pcschools.us/woad-
local/users/mpurzycki/ PCR_powerpoint_review.pdf. Retrieved on 08 January 2011.
Polymerase Chain Reaction: Chromosome 16/Alu-tpa25. Retrieved from
http://www.hanoverhigh.us/ departments /science/rm215/genetics/pdf/labs/pcr_alu.pdf.
Retrieved on 08 January 2011.
Polymerase Chain Reaction Preparation & Amplification. Retrieved from
www.docstoc.com/docs/.../ The-Polymerase-Chain-Reaction-gel. Retrieved on 08
January 2011.
Primer Design. Retrieved from http://bioweb.uwlax.edu/GenWeb/Molecular/seq_anal/primer_
design/primer_design.htm. Retrieved on 09 January 2011.