HUMAN GENOMIC DNA EXTRACTION AND POLYMERASE CHAIN REACTION

(PCR) AMPLIFICATION OF ALU PV92

GROUP MEMBERS
NAME MATRIC NUMBER

GROUP 2 (KSA)
LECTURE’S NAME: DR MARYAM BINTI MOHD REHAN AND DR NAZMI BIN
ABDUL MANAP
SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR
MOLECULAR BIOLOGY (SBP 3053)

15 NOVEMBER 2019
2.0 Introduction

Human genome consists of 2.9 billion base pairs of DNAs. From this total, only 5% consists
of exons which code for protein. Introns and other noncoding sequence make up the
remainder; although some of these sequences possess undiscovered functions, most of it
appear to have nonfunctional properties. Most of these noncoding sequences always self-
replicating and are repeated for thousands of times in the genome and thus, account for more
than 20% of human genome.

Although DNA from any two people is more alike than different, many chromosome
regions exhibit sequence differences between individuals. These variable sequences are
termed as “polymorphic” which are widely used in the study of human evolution, as well as
for disease and identify testing. Most of the polymorphism are in the estimated 98% of the
human genome does not encode protein.

In 1979, it was discovered that human DNA contains 300 base pairs repetitive
element. Copies of this element contain a recognition site for the restriction enzyme Alu 1,
and were subsequently named Alu elements. Alu elements are classified as SINEs, or Short
Interspersed Elements. All Alu elements are approximately 300-bp in length and their names
are derived from a single recognition site for the endonuclease Alu 1, located near the middle
of Alu sequence. Human chromosomes contain about 1,000,000 Alu copies, which equals to
10% of the total genome. Based on scientists, an estimated around 500-2000 different Alu
elements are found scattered across the human genome. Although Alu elements have been
found in exons, but most of it exists in intron and other non-coding regions. Interestingly,
introns often vary in their size and sequence among individuals while exons do not. This
variation is the result of differential accumulation of mutations in DNA throughout evolution.
We do not notice the mutations in non-protein coding regions because they do not affect our
phenotypes. However, these differences represent the molecular basis of DNA fingerprinting
used in human identification and studies in population genetics.

PCR or known as Polymerase Chain Reaction was invented in 1984 by world

renowned scientist, Kary Mullis who was awarded a Nobel Prize for his work in 1994. The
enormous utility of PCR is based on its ease of use and its ability to amplify DNA. PCR has
an impact on four main areas of biotechnology: gene mapping, cloning, DNA sequencing and
gene detection. PCR is now used in medical fields as a medical diagnostic tool to detect
specific mutations that may cause genetic disease, in criminal investigations and courts of law
to identify suspects on a molecular level, and as a powerful tool in the sequencing of the
human genome.

The objective of PCR is to produce a relatively large amount of a specific piece of

DNA from a very small amount. By means, the controlled enzymatic amplification of a
template DNA molecule containing a specific DNA sequence of interest. The template can be
any form of double stranded DNA such as genomic DNA. A trace of amount of DNA from
drop of blood, a single hair follicle or cheek cell and use PCR to amplify millions of copies of
the desired DNA fragment. The process of PCR consists of the three major steps; denaturing
the sample DNA which separates the double-stranded DNA with heat, annealing in which the
binding of the primers with the single stranded DNA template occurs and extending that
extends the primer with the help of DNA polymerase resulting in the double-stranded
molecule.

In order to perform PCR, ones must know which sequence region that they wished to
amplify so that they can make specific primers to conduct the PCR amplification. A primer is
a short piece of single-stranded DNA (17-40 bp) that is manufactured in a laboratory. 2
primers are needed for each PCR reaction. Primer 1 will hybridize to a sequence at the 5’ end
of the target sequence, at the 5; end of one strand at the genomic DNA, and the second
primer, primer 2 will hybridize to the 3’ end of the target sequence, at the 5’ end of the
complementary strand of DNA. In the first step of PCR the genomic DNA is totally
denatured by heating it at a high temperature. Then, the sample is cooled down to allow
primers to anneal or hybridize to their complementary sequences. The temperature for the
hybridization step of PCR depends on the sequence and length of the primers being used. The
hybridization step may be carried out between 42-62 degree celcius depending on the primers
used. The last step in PCR reaction is the extension step. In this step, DNA is synthesized
from the primers, using the target DNA as a template. DNA synthesis is performed using a
special DNA polymerase called Taq polymerase that is active at high temperatures. Taq
polymerase is a component that is isolated from a bacterium that lives in hot springs called
Thermophilus aquaticus. The optimum temperature for Taq polymerase is around 70 degree
celcius,but it is also stable at more higher temperatures. DNA synthesis by Taq polymerase,
as for all DNA polymerases, requires a primer, a DNA template, the nucleotides dATP,
dGTP, dCTP, and dTTP and Mg+. in extension step, the length of primer is extended by
addition of nucleotides during DNA synthesis. Since DNA is being synthesized from both
strands of DNA, two different primers are needed, one for each strand.
 Human DNA is composed of a great number of repeated sequences which represent
more than 50% of the genome. Alu sequences are one of the most abundant repetitive DNA.
In this experiment, each student is hunting for Alu element by extracting his/her DNA from
hair follicle cell that is located on chromosome 16 at the PV92 locus by PCR. As a control,
DNA purified from a cultured hair cell line may be used. This particular Alu element is
dimorphic which mean that the element is present in some individuals and not others. This
Alu sequence is 300 bp long. Some of these Alu sequences have characteristics that make
them very useful to geneticists because these polymorphic sequences can provide the basis
for genetic disease diagnosis, forensic identification, and paternity testing. The PCR
product(s) will then be examined on agarose gels and some people can observe that they have
the Alu sequence in one copy of their 16 th chromosomes (one allele) (+/-) where we called
them as heterozygous of Alu sequence, others may have the homozygous in having Alu
sequence in both copies of their 16th chromosome (+/+),, while others may not have the insert
on either copy of the 16th chromosome (-/-) where we called them as homozygous without
Alu sequence. Objectives of this experiment are the isolation of human DNA and the
comparison of DNA polymorphisms between individuals by PCR amplification and agarose
gel electrophoresis.

3.0 Methods

Isolation of DNA from human hair

Firstly, one screwcap tube containing instagene matrix and protease solution is
labelled. Next, isolate 2 hairs containing a sheath, a barrel-shaped structure often white in
color, encircling the shaft near the roots of the hair. The collected hair is soaked in the
labelled screwcap tube containing 200 microlitre instagene matrix and protease. Then, the
labelled tube is incubated for 10 mins at 56 degree celcius. After 5 minutes, the labelled tube
is being vortex and re-incubate for another 5 minutes. After that, the tubes is removed and
being vortex once again before being placed in the 100 degree celcius heatblock for 5
minutes. Then, the tube is removed from the heatblock and cooled for 2 minutes before being
vortex again. The tube is centrifudged using spin microcentrifudge for 30 seconds. Carefully
remove the 50 microlitre of supernatant and transfer to a clean 0.5 mL microcentrifudge tube.
The tube with pellet is discarded. The tube containing supernatant is placed on ice.
Amplification of the PV92 Locus

The PCR reaction is conducted by labelling the 0.2mL PCR tube. Then, the PCR tube
is placed into the capless micro test tube and being hold by the foam micro test tube holder.
20 microlitre of supernatant with genomic DNA is transferred into the bottom of the PCR
tube. Next, 20 microlitre of complete PCR Master Mix is added into the PCR tube containing
DNA samples. The Master Mix contains 2 different primers, dNTPs, Taq polymerase and
MgCl2. The mixture is mixed by pipetting up and down. The capped tube is then ready to be
used in the PCR thermocycler. Thermocycler is a device that is used to conduct the PCR
reaction. It is an automated process that have cycle in different temperatures. The
thermocycler is made up of aluminium and gold block which functions as a good heat
conductor that can hold up to 100 degree celcius at one time. There are about 64 samples that
can be filled up in thermocycler at one time to reduce time consumption and many samples
can be carried out in one time. The lid of the devices can be heated up to 105-degree celcius
which is to prevent the condensation or evaporation process from takes place during the PCR
reaction is being conducted. The following programme will be used:

Step 1: pre-denaturation process; where the sample is heated for 2 minutes in 94-
degree celcius

Step 2: denaturation process; where the sample is heated up to 94-degree celcius in 1

minute.

Step 3: hybridization process; where the sample is hybridized in 60-degree celcius for
1 minute

Step 4: extension process; where the sample is extended in 72-degree celcius in 2

minutes

(Step 1-4 is repeated for 40 times)

Step 5: final extension process; the sample is heated up to 72-degree celcius for 10
minutes.

Step 6: the sample is hold up at 4-degree celcius.

Then, the PCR samples is removed from the thermacycler. The PCR tube is centrifudge for 3
seconds on the maximum rpm to separate completely the mixtures based on their weights.
Electrophoresis steps of amplified samples

As for the electrophoresis, firstly, agarose gel of 1.0% was prepared. The solidified gel placed
in an electrophoresis chamber and 0.25X TAE buffer was poured to cover the gel. Then, the
PCR samples were centrifuged for 3 seconds at 12000 rpm. 2 microliter of PV92 XC loading
dye was dropped on a piece of Parafilm and followed by adding 10microliter of the sample
and mixed well on the parafilm before loading it in the well of the gel. To load the sample
into the well of the gel, micropipette was used. At the first lane, the lab instructor load DNA
size markers into the first well.So that, the bands of the markers will be used to estimate the
sizes of the PCR products. The electrophoresis switched on and was run at 200 volts for
about 20 minutes, until the migration of the blue dyes can be observed.

Staining of agarose gel

After electrophoresis completed, the gel tray and the gel itself removed. In order to stain the
agarose gel to allow us to see the band, 120 ml of 100X Fast Blast DNA stain into the
staining tray and let the gel stained for about 2 minutes with gentle agitation. Then, the gel
was rinsed with warm tap water for approximately 10 seconds. After that, the results can be
observed under the light background to see the bands clearly.

4.0 Result and discussion

The presence or absence of this insert or known as Alu sequence can be detected using the
polymerase chain reaction followed by agarose gel electrophoresis. Since we are amplifying a
region of DNA contained within an intron, the region of DNA is never really used in our
body because our body normally will spliced off the intron since it is a non-coding region.
So, if someone don't have it, don't worry. the primers in this kit are designed to bracket the
region within the PV92 region that is 641 base pairs in length if the intron does not contain
the Alu insertion or 941 base pairs in length if Alu is present .This increase in size is due to
the 300 base pair sequence contributed by the Alu insert.
Gel electrophoresis is one of the major methods utilized in molecular biology for the
analysis of DNA. This method involves the migration of fragments of DNA through a gel,
where they are separated based on size(molecular weight) or shape. Gel electrophoresis
involves the use of a gel usually made out of polymers such as agarose. The gel is immersed
in a buffer solution that conducts an electric field. The DNA sample of interest is first
fragmented using restriction enzymes and is then injected into the gel. When the electric field
is turned on, the DNA fragments which have the nucleotide is negatively charge in the gel
and it will migrate toward the positive electrode. If the DNA fragments are of different sizes,
then the migration times will be different for each size fragment. The matrix of the agarose
gel acts as a molecular sieve through which smaller DNA fragments call move more easily
than larger ones. The difference the percentage of the agarose gel, the difference the time
taken for the band to migrate because of the higher density of the molecular sieve. The
fragments are then visualized using a dye or autoradiography and are visible as bands in the
gel.
A molecular-weight size marker,
also referred to as a protein ladder, DNA
ladder, is a set of standards that are used
to identify the approximate size of a
molecule run on a gel during
electrophoresis, using the principle that
molecular weight is inversely
proportional to migration rate through a
gel matrix. It is produced through the
use of restriction enzymes and a
recognized DNA sequence. The DNA is digested by a particular restriction enzyme, resulting
in DNA pieces of varying molecular masses. On the other hand, the size of the DNA pieces
are based on the sites where the restriction enzyme cuts. Thus, easier for the experiment to
compare their molecule with DNA ladder to know the size in base pair units.
When the PCR products are
electrophoresed on an agarose gel,
there are three distinct outcomes
that can be visualized. If both
chromosomes contain Alu inserts,
then each amplified PCR product
will be 941 base pairs long. On a gel
these will migrate at the same speed
so there will be one band that
corresponds to 941 base pairs. If neither chromosome contains the insert, then each amplified
PCR product will be 641 base pairs and they will migrate as one band that corresponds to 641
base pairs. If you have an Alu insert on one chromosome but not the other, then there will be
one PCR product of 641 base pairs and one of 941 base pairs.The resulting gel will reveal
two bands. In our experiment only three of us showing the results where only one band where
when compared to the ladder,it had migrate near to the well. This showing that three of us
have Alu sequence with base pair of 941 for both chromosomes respectively. Meanwhile, for
the other member who her result are not showing,it might be because of the concentration of
DNA(PCR product) loaded might be very less or very high or the hair sheath sample that she
took does not contain the epithelial cell.

5.0 Conclusion

In a nutshell, PCR is a technique used to amplify or copying certain part of DNA which in
this lab is at PV92 gene. This experiment is conducted to study the isolation of human DNA
and the comparison of DNA polymorphisms between individuals by PCR amplification and
agarose gel electrophoresis. Alu element is a component that resides in non-coding region of
DNA which have no functional properties on phenotypes but it is commonly used to
differentiates between individuals in terms of fingerprints and diseases. In this experiment,
we are trying to find out about the differences characteristics between students based on the
Alu element on chromosomes 16 in locus PV92. Then, the genomic DNA is amplified using
PCR by using thermocycler to produce double stranded DNA and the sample is analysed
using gel electrophoresis to have the definite results that determine the characteristics among
individuals.

References:

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