Table of contents

Page Number

Introduction ………………………………………………………………… 2

Materials ……………………………………………………………………. 5

Methodology………………………………………………………………… 7

Results……………………………………………………………………….. 12

Discussion…………………………………………………………………… 15

References..…………….………………………………………………………21

Appendix. .…………………………………………………………………….. 25

1
1. Introduction
DNA is a polymer of deoxyribonucleoside covalently linked by 3´-5´ phosphodiester bonds.
It exists as a double stranded molecule, where the two strands coil around each other forming
a double helix. Each polynucleotide strand is a polymer of nucleotides. A nucleotide consists
of a deoxyribose sugar, nitrogenous base and a phosphate group. Nitrogenous bases are
classified into purines and pyrimidines. Purines consist of a double ring structure and
pyrimidines consist of a single ring structure. Adenine and guanine are purines and cytosine
and thymine are pyrimidines. DNA is stabilized by hydrogen bonds formed between the bases.
Complimentary base pairing occurs between adenine and thymine by the formation of two
hydrogen bonds and three hydrogen bonds between cytosine and guanine. The two
polynucleotide strands are parallel but run in opposite direction hence they are referred as
anti-parallel strands. Due to base pairing, polar sugar phosphate backbone exposes outwards
and non-polar base pairs are extended into the interior surface of the helix. This makes DNA
water soluble (Taylor, Green and Stout, 2008). In eukaryotic cells, DNA is associated with
nucleoprotein present in the nucleus whereas in prokaryotes the protein- DNA complex is
present in a non-membrane bound region known as the nucleoid (Chandar and Viselli, 2010).
Functions of DNA include replication of DNA where it plays a role in transmitting hereditary
information from the parent to the offspring and biosynthesis of RNA and proteins. DNA can
be extracted from blood, hair roots, buccal cells, saliva and semen (Alberts et al, 2002).

One of the most common ways to obtain human blood is through Venipuncture.
Various methods have been used to extract DNA from blood. In most of these methods, toxic
organic solvents and enzymes are used. Since, these enzymes are not quite affordable for
everyday research work and the other materials used are toxic. Therefore, genomic DNA was
extracted using the salting out method. This method makes use of high salt conditions to
precipitate proteins leaving the DNA in the solution. DNA is then precipitated using alcohol or
isopropanol (Nasiri et al, 2005). DNA extraction from human blood using salting out method
is done in three basic steps as in Cell lysis, Protein digestion and precipitation and DNA
isolation (Barbosa and Chaves, 2016).

To separate components of a complex mixture using a centrifuge is referred to as

centrifugation. By the application of centrifugal force, a centrifuge is used to separate two
miscible substances causing the migration of denser components away from the axis of rotation
and migration of the less dense components towards the axis of rotation. The particles that
precipitate at the bottom is called the pellet and the remaining solution or the isolated specimen
known as the supernatent can be further processed. There are two types of centrifugation they
are, preparative and analytical centrifugation. A preparative centrifuge is used to pellet small
particulate matter such as viruses, organelles and nucleic acids and an analytical centrifuge is
used to examine the mass of macromolecules and proteins. Based on the samples and
experiments different types of rotors are used in the centrifuge such as fixed angle rotors,
Horizontal rotors and Swinging bucket rotors (Smith, 2013).

2
In the following practical centrifugation is necessary to separate the dissolved DNA from the
cell debris after cell lysis and it also aids the isolation of DNA from protein debris and salts
added.

Agarose gel electrophoresis is a technique used for the separation of proteins, DNA and RNA
based on their size, shape and charge under the influence of an electric field. Movement of
nucleic acid fragments across the Agarose gel occurs when an electric current is supplied. As
DNA consists of a negatively charged backbone it will migrate from cathode to anode.
Movement of nucleic acids is influenced by several factors such as:

 Size of the fragment

 Confirmation
 Charge applied
 Concentration of Agarose
 Size of the fragment

Agarose gel is a polysaccharide matrix that works like a sieve. Molecular sieving can be
referred to as higher the concentration of the gel smaller the pore size and lower the
concentration larger the pore size. Thus, size of the pores can be altered by changing the
concentration of the gel. Moreover, smaller fragments move faster than larger fragments, super
coiled DNA moves faster than linear DNA, Higher the voltage faster the movement of
fragments but increased voltage can melt the gel (Lee et al, 2012), (Voytas, 2001).

Figure 1. Running of gel electrophoresis (Rogers, 2017)

3
The obtained DNA sample then undergoes PCR amplification. Polymerase chain reaction
(PCR) is a technique used to synthesize millions of copies of a targeted DNA sequence.
In this practical, PCR is carried out to amplify exon 5 of p53 gene using a p53 primer.
PCR occurs in three main steps:

1. Denaturation
2. Annealing
3. Extension

Denaturation involves the separation of strands, Annealing is the binding of the reverse and
forward primers to the 3´ end of the template and Extension is the process where DNA
continues to synthesize on both strands. The occurrence of these steps is referred to as one
cycle. In order to amplify DNA 30-40 cycles need to take place (Butler, 2012). Thereafter,
verification of PCR products is done on Agarose gel and to compare the size of DNA
fragments, a ladder with a mixture of fragments with known size is introduced (Kavya, 2015).

Figure 2. Basics of PCR cycling (Kavya, 2015)

2. Objectives
 Objective 1: Extraction of genomic DNA from human blood
 Objective 2: Quantification of DNA using Agarose gel electrophoresis
 Objective 3: Amplification of p53 genome using Polymerase chain reaction (PCR)
 Objective 4: Observation and quantification of PCR products

4
3. Materials
Equipment Reagents Samples Other
3.1 Venipuncture
Falcon tubes Isopropyl 70% alcohol Human blood Cotton
Needles Gauze
Syringe Gloves
Swab
Tourniquet
3.2 DNA extraction

Autoclave 70% Ethanol Human DNA Gloves

Centrifuge 100% Ethanol
Eppendorf tubes EDTA
Pipettes Cold lysis buffer
Vortex Proteinase K
100 %Sodium Dodecyl
Sulfate
Saturated NaCl
SE buffer
TE buffer
Tris
3.3 Quantification of extracted DNA by Agarose electrophoresis
Electrophoresis Agarose powder Group 1A DNA Gloves
tray sample
Fume hood DNA markers of Group 1B DNA Parafilm
 25ng/µl sample
 50ng/µl
 100ng/µl
Gel documentation Ethidium Bromide Power supply
system
Gel cassette PCR buffer
Gel box 1x TAE buffer
Gel lid
Microwave
Micro pipettes

5
Equipment Reagents Sample Other
3.4 Amplification of DNA by PCR

Eppendorf tubes Autoclaved distilled water Group 1A DNA Gloves

Micro pipettes DNA Group 1B DNA
PCR dNTP
Forward Primer
MgCl2
PCR buffer
Reverse primer
Taq

3.5 Running and quantification of PCR

Electrophoresis Agarose powder Group 1A DNA Gloves

tray sample
Gel cassette 100 base pair ladder Group 1B DNA Power supply
sample
Gel documentation 1x TAE buffer
system
Gel lid
Gel box
Micro pipettes
Microwave

6
4. Methodology
Glassware, Eppendorf tubes, micropipette tips and solutions were sterilized by autoclaving at
121o C for 15 minutes and all except solutions were dried overnight in a hot air oven before
use. General reagents and composition of commonly used solutions and buffers are detailed in
the appendix. All chemicals were of analytical grade. All enzymes were obtained from
Promega (USA) and Amersham (Sweden) and were used according to the manufacturer’s
instructions.

4.1 Venipuncture

A volunteer came up and was kept to be seated comfortably in a chair with the arm rested and
extended on the table to form a straight line from the shoulder to the wrist. The vein of choice
was the median cubital vein. The place to be injected was cleaned with a swab containing
the antiseptic agent (isopropyl alcohol 70%) in circular motion. Thereafter, the limb was held
with the non-dominant hand and the vein was anchored with the thumb.
The dominant hand then positioned the needle bevel side up at 45º angle to skin surface.
Thereafter, the needle was entered in a quick, sharp, darting motion and decreased the angle to
15º, entering the vein the tourniquet was released. The plunger was then pulled back slowly to
aspirate blood. The gauze pad was held with pressure over the injected site and the needle was
pulled straight from the vein. The gloves, needles and gauze were discarded in a proper manner.

4.2 DNA extraction

Firstly to lyse the cell, the blood was transferred into a falcon tube and 7.5 ml of cold lysis
buffer was added. It was then incubated for 15 minutes in crushed ice and centrifuged for 15
minutes at 3000 rpm. The supernatent was then discarded and 2.5 ml of SE buffer was added
to the pellet and vortexed. And then again the tube was centrifuged for 15 minutes at 3000 rpm
and the supernatent was discarded. To the pellet, 1.5 ml of SE buffer was added and vortexed
and thereafter, 0.125 ml of 100% SDS and 0.007 ml of Proteinase K were added to the sample.
The tube was then left to incubate overnight at room temperature. For the digestion and
precipitation of protein, 0.5 ml of saturated NaCl was added to the sample and shook
vigorously. The sample was then centrifuged for 15 minutes at 3000 rpm and the supernatent
was transferred into a falcon tube. The volume of the supernatent obtained was 5 ml. Thereafter,
the precipitated pellet of proteins were discarded. To isolate DNA, Added 10 ml of cold 100%
ethanol to the supernatent and inverted the tube as shown in figure 9. The obtained DNA was
then transferred into an Eppendorf tube and was washed several times with 70% ethanol. The
DNA was then left to air dry and an appropriate amount of TE buffer was added to dissolve
DNA. The sample was then incubated overnight at -4℃ and stored at -20℃ for long term
storage.

4.3 Preparation of 0.8% Agarose gel

0.4g of Agarose powder was mixed with 50 ml of 1x TAE buffer solution and heated
(Microwaved) till all Agarose got dissolved. The solution was then kept to cool down inside
the fume hood, and to it 2.8µl of Ethidium Bromide was added.

7
4.4 Preparation of loading samples (DNA samples)

On a piece of parafilm, pipetted 7µl of PCR buffer for each sample. And onto it, 1 µl of 25 ng,
50ng and 100ng were added. The samples were then pipetted in and out to ensure proper
mixing.

Figure 3. Preparation of gel loading samples on to a parafilm (The Friedman lab chronicles,
2010)

25ng 50ng 100ng GR.1A GR.1B

Figure 4. Order of samples on the parafilm

4.5 Running of Agarose gel

The gel cassette was then set and the dissolved Agarose which had been cooled was poured on
to it in a way where no air bubbles were created and the setup was left to solidify as shown in
the diagram below.

Figure 5. Solidification of Agarose gel (Wikipedia, 2017)

8
The comb was then removed carefully creating wells and the gel was placed inside an
electrophoresis tray. 1x TAE buffer was then added till it reached approximately 3-4 ml above
the gel .The wells were then loaded with the known markers and group 1A and 1B DNA
samples as shown in the diagram below.

Figure 6. Loading the samples into wells (Shutterstock, 2003)

Closed the lid of the electrophoresis tray and plugged in the electrodes to the correct slots. The
DNA was then left to run under 50 V and the result was visualized using a gel documentation
system. And this way the concentration of DNA was found and the relevant sample was further
used for PCR amplification.

1 2 3 4 5

Figure 7. Order of loading the DNA Samples

Table 1. Concentration of DNA

Number Concentration (ng/µl)

1 25
2 50
3 100
4 Group 1A sample
5 Group 1B sample

9
4.6 Amplification of DNA by polymerase chain reaction

The PCR components were first towed and a spin was given. Thereafter, a PCR master mix
was prepared by the addition of the components and their respective volumes as given in table
2 into a reaction tube and a control. Then the mixture in the tubes were topped up to 25µl by
the addition of autoclaved distilled water.

Table 2. PCR components and their concentrations

Components Stock Volume Final

concentration concentration
Reaction tube Control
MgCl2 25mM 1.5 µL 1.5 µL 1.5mM
PCR Buffer 5x 5 µL 5 µL 1x
dNTP 10mM 0.5 µL 0.5 µL 0.2mM
Forward primer 2µM 2.5 µL 2.5 µL 0.2mM
Reverse primer 2µM 2.5 µL 2.5 µL 0.2mM
Autoclaved 11.75 µL 12.75 µL
distilled Water
DNA 100ng/ µL 1 µL -
Taq 5U/µL 0.25 µL 0.25 µL 0.05U/µL

The DNA samples were then briefly centrifuged to collect all the drops from the walls of the
tubes and put into the PCR machine for the amplification of DNA, which is the exon 5 of p53
gene. This amplification followed the PCR cyclic conditions given in table 3.

Table 3. Thermal cyclic conditions

Step Temperature Time

1. Initial denaturation 94 ℃ 4 min
2. Denaturation 94 ℃ 1 min
3. Primer annealing 58 ℃ 1 min
4. Extension 72 ℃ 2 min
5. Final extension 72 ℃ 11 min

The extension step consisted 35 cycles of amplification and upon completion it moved onto the
final extension.

4.7 Preparation of 1% Agarose gel

0.5g of Agarose powder was mixed with 50 ml of 1x TAE buffer solution. It was then
microwaved till all the Agarose got dissolved. The Agarose solution was then kept to cool
down inside the fume hood and to it 2.8µl of ethidium bromide was added.

10
4.8 Running and quantification of PCR products

Repeated the procedure as in 4.5, but here the wells were loaded with 4µl of a 100 base pair
ladder and 8µl each of the Group 1 A and B samples and their respective controls. The setup
was then closed and the electrodes were plugged to the correct slots and left to run at 55V. The
result was then visualized using a gel documentation system and the band size of DNA in the
samples were found.

1 2 3 4 5

Figure 8. Order of loading the samples

Table 4. Indication of the Sample name and type

Number Sample name

1 100 base pair ladder
2 Group 1A sample
3 Group 1A control
4 Group 1B sample
5 Group 1B control

11
5. Results
5.1 DNA extraction

Precipitation of DNA

Figure 9. Precipitation of DNA

Figure 10. Precipitated DNA when washed with 70% Ethanol

Figure 11. Precipitated DNA

12
5.2 Quantification of DNA using Agarose gel electrophoresis

Dye front

Figure 12. Running the DNA samples in 0.8% Agarose gel

Dye front

Figure 13. Results of 0.8% gel electrophoresis DNA samples when viewed through a gel
documentation system

Here Group 1A DNA sample has the same intensity as the 100ng/µl by which we can
conclude that concentration of 1A is 100ng/µl.

Group 1B has a thick white band and the reason why it had occurred will be discussed below
under discussion.

13
5.3 Observation and quantification of PCR products

200 bp

Figure 14. Results of 1% Agarose gel electrophoresis when viewed through the gel
documentation system

The Group 1A and B samples show an approximate band size of 200 base pairs and the
negative controls here show no bands which indicates that there had not been any form of
sample contamination.

14
6. Discussion
Acquiring high quality intact DNA is the most critical step in many fundamental molecular
biology applications such as DNA cloning, sequencing, PCR and electrophoresis (Alberts,
2002). In the above practical DNA was extracted from Human blood which was obtained
through venipuncture. Venipuncture is a process where a particular vein is punctured to draw
blood for medical purposes and Venipuncture site is where that vein is located (Genden, 2017).
So in order to obtain blood, Venipuncture was done in a volunteer. The most commonly used
site for drawing blood is the median cubital vein located at the bend of the elbow. The reason
why most phlebotomists use the median cubital vein is that it lies close to the surface of the
arm and becomes prominent when pressure is applied. Still, phlebotomists must be careful
when drawing blood from this area as it poses a major health risk when punctured deeply
(WHO, 2010). The median cubital vein also connects the cephalic vein and the basilic vein
which serve as Venipuncture sites. The cephalic vein is only used as a venipuncture site when
the median cubital vein is hard to see and feel. The tip of an injection needle is bevelled as it
helps cut through the skin with minimum trauma. Generally, the needle is kept in the bevel up
position in so that the needle slices the skin and not tears it. This causes less pain compared to
the bevel down position (WHO, 2010). Blood can be collected using different anticoagulants
including citrate, ethylenediaminetetraacetic acid (EDTA) or heparin (Bowen and Remaley,
2014). Kotikalapudi and patel (2015) said Results from blood DNA isolation can be affected
by the type of anticoagulant used and this in turn can influence the diagnostic tests associated
with blood.

EDTA is the anticoagulant of choice for blood collection for DNA extraction. Hence, we use
EDTA coated tubes as EDTA is a chelating agent and binds calcium ions in blood which is
essential for the clotting mechanism thereby, preventing coagulation. Cold Lysis buffer is a
hypotonic solution containing NH4Cl, EDTA and KHCO3. Red blood cell membranes are
effectively permeable to NH4Cl and cell lysis occurs due to the unbalanced osmotic pressure
as a result of the movement of water from the exterior to the interior of the cells (Phillips,
Hosking and Shelton, 1983). The lysis of the red blood cells causes the solution to become red
in colour as haemoglobin is released. The DNase I is the major nuclease present in blood and
it degrades DNA. The cold condition is maintained to inhibit DNase enzyme activity by the
anticoagulant EDTA which is an indirect DNase I inhibitor, since it chelates divalent ions such
as calcium and magnesium which are essential for the enzyme structure and efficient enzymatic
activity, it deactivates the enzyme activity ensuring the protection of the circulating cell free
DNA from degradation in blood samples by the nucleases (Barra and Costa, 2015). Incubation
provides sufficient time for the lysis of the RBC (Ghatak, Muthukumaran and Nachimuthu,
2013).

Centrifugation is the process by which a centrifuge can spin up to facilitate the separation of
the phases of DNA extraction (Smith, 2013). . Here, upon spinning at 3000rpm for 15 minutes
the supernatant and pellet were formed. Upon addition of the cold lysis buffer and SE buffer
the supernatant was discarded but upon addition of sodium chloride the pellet was discarded
as the supernatent consisted of the extracted DNA sample. SE buffer contains NaCl, EDTA
and Tris.
15
It washes the white blood cell pellet and provides appropriate pH required for proteinase K
action (Ghatak, Muthukumaran and Nachimuthu, 2013). Vortexing is often effective for
breaking the pellet which in turn increases the surface area and enables efficient washing upon
the addition of SE buffer.

Sodium Dodecyl Sulfate (SDS) which is a detergent degrades white blood cells and its nuclear
membrane releasing both its proteins and DNA to the exterior. Proteinase K is added to
breakdown proteins by cleaving the peptide bonds adjacent to the carboxylic group of amino
acids. It also degrades nucleases or enzymes that may be present in the sample. This is
important since these chemical compounds can attack and destroy the DNA in the sample.
Addition of saturated NaCl binds with water as the salt disassociates into ions and they form
interactions with water reducing the availability of water to form interactions with proteins.
Therefore, occurrence of protein- protein interactions reduces the solubility and causes the
precipitation of proteins. Usually after the addition of saturated NaCl to the sample and
centrifuging it a colourless supernatant and a white pellet is formed. But in the experiment we
conducted both the phases were slightly pink in colour which signifies that all red blood cells
had not been lysed. So in order to overcome this we could carry another step of addition of
lysis buffer. Since, DNA is insoluble in ethanol, the addition of cold ethanol will cause the
DNA to precipitate as because ethanol has a low dielectric constant than water it promotes
ionic bond formation between Na+ from the salt and PO43- from the DNA backbone. Also as
the solubility of DNA is low at low temperatures, cold conditions facilitates precipitation of
DNA. Cold ethanol is added twice the volume of the supernatent.

That is;
volume of supernatent obtained = 5 ml
Therefore, added cold ethanol = 10 ml
Upon addition of ethanol the tube is tilted so as to obtain more DNA as shown in figure 9.

70% ethanol is added to wash away the remaining salt and finally, Tris.EDTA (TE) buffer
dissolves DNA and prevents degradation of DNA by maintaining a proper pH. The obtained
sample was then stored at -20℃ for long term storage (Ghatak, Muthukumaran and
Nachimuthu, 2013), (Chacon-cortes, 2014).

Group 1A did not obtain appropriate results as the pellet containing the DNA had been
discarded in one of the above steps. In order to avoid this, the supernatent should be carefully
spilt without tilting the tube backwards as it could drain away the pellet too. In order to
prevent sample contamination careful handling of biological material is essential (Elkins,
2013). Not touching the exposed needle during Venipuncture, Working in a sterilized
laboratory, Pipetting in the appropriate manner, Usage of EDTA tubes and not heparin coated
tubes as it interferes with the purity of DNA and it also inhibits downstream PCR analysis,
Storing DNA samples to sunlight and/or temperatures above 4℃ should be minimized to
preserve the DNA in the sample are possible ways to avoid contamination (Rice, 2017).

16
DNA extraction has developed into a diversity of laboratory techniques. The currently available
methods are: DNA extraction methods using magnetic beads, Solid – phase DNA extraction
methods, DNA extraction methods using silica and silica matrices, Solution- based DNA
extraction methods using salting out, DNA extraction using anion exchange resins and Solution
based DNA extraction methods using organic solvents. DNA extraction has evolved from
solution and solid-phase manual techniques initially performed manually before incorporating
these into automated methods. As all these methods differ in many different aspects it is very
difficult to determine the best choice available. Generally, an extraction method is chose based
on the availability of equipment, samples, reagents, as well as considering speed, extraction
efficiency and quality, technical requirements and cost (Chacon-cortes, 2014).

Agarose was microwaved as it is a polysaccharide and needs to be heated for it to dissolve. It

solidifies below 42 ℃. 0.8% Agarose gel was prepared as because the concentration affects the
size of pores. Higher the concentration smaller the size of pore and smaller the concentration
higher the size of pore. For the movement of genomic DNA through the matrix larger pores
were needed as it is bulky and has a higher molecular weight, so the concentration was made
up to 0.8%. Movement of DNA towards the anode occurs due to the negative charges of the
phosphate backbone (Lee et al, 2012). Ethidium Bromide (EtBr) is added to visualize DNA. It
is an intercalating agent and binds to DNA and emits fluorescence when exposed to UV light
as shown in the figure. As seen in the figure the band that has a colour in it is the dye front.
The loading buffer that was mixed with the DNA sample usually has a gel loading dye in it and
bromophenol blue is the commonest one. Orange G, xylene, cyanol, cresol red are examples
for loading dyes. It typically migrates through the gel faster than the DNA enabling the tracking
of the molecules. It does not absorb UV light hence no fluorescence is emitted. EtBr should be
handled with care as it is highly carcinogenic (Sigmon, 1996). Conductance of current results
in electrolysis of water which may cause the release of H+ ions. These H+ ions can bind with
the negatively charged DNA and neutralize it which may result in change in pH therefore, TAE
was used as the electrophoresis buffer to maintain appropriate pH and prevent the alteration of
charges of nucleic acids. Also, Ions in the buffer facilitate the conductance of current. The
commonly used buffers are Tris/acetate/EDTA (TAE) and Tris/borate/EDTA (TBE). TAE has
a low buffering capacity and gives a good resolution to long nucleic acid fragments whereas
TBE has a high buffering capacity and gives a good resolution for small nucleic acid fragments.
TBE has a higher ionic strength than TAE, therefore when added TBE the DNA migration is
high as higher the ionic strength higher the electrical conductance (Lee et al, 2012), (Voytas,
2001). DNA markers were added in the concentration of 25ng/µl, 50ng/µl and 100ng/µl in
order to find the intensity of the sample 1A and 1B. As shown in the figure 13, sample 1A has
the same intensity as the third DNA marker whose concentration is 100ng/µl and the sample
1B has a thick white band from which we can conclude that it has a higher intensity than
100ng/µl. This is because it contains undissolved DNA which can occur due to pipetting from
the bottom of the vial containing the DNA sample. This results in high thickness and cause
difficulty in separation of DNA of different lengths. This can be resolved by further dilutions
and the intensity can be determined by carrying out further experiments. The concentration of
the sample 1B was found to be 720 ng/µl when measured through a photo spectrometer. The
sample 1A was further used for PCR amplification.

17
Initially the PCR components were thawed on the work bench as they were stored inside the
refrigerator. A PCR mixture was prepared by the addition of several components and was
topped up to 25µl by adding autoclaved distilled water and not PCR water as it is expensive
and is used for sensitive practical like research work. So to cut down cost autoclaved distilled
water was used. All components were maintained in cold conditions to protect the samples and
avoid thermal degradation. Since, PCR is sensitive a negative and positive control are used.
The positive control contains the PCR mix with the DNA and the negative control contains the
PCR mix without the DNA.

A negative control is used to ensure the experimental results are free of contamination and non-
specific amplification. Usually a band does not appear in a negative control but if it does then
there is some form of contamination. If the bands in the negative control are smaller than the
sample in the positive control then it could possibly be a primer dimer but if the band size is
similar to the positive control then it means there is some template contamination. This could
occur if any of the reagents are contaminated or it could possibly be the pipette tips as well. To
re setup the reaction using new reagents would be the simplest solution but if needed to find
the source of contamination then you would need to change a reagent one at a time. Filter tips
could be replaced if the pipette is the source of contamination. Since, filter tips are expensive
cleansing the pipette well would also be appropriate.

To begin with each component and its purpose, Addition of autoclaved distilled water provides
the liquid medium for the reaction to occur. PCR buffer and MgCl2 are important for the
functioning of the Taq DNA polymerase. Mg2+ ions in MgCl2 acts as a coenzyme factor and
binds with primers, templates and dNTPs resulting in the formation of complexes. A decreased
amount of Mg2+ ions can result in a low yield of PCR product and a higher amount can give
rise to non-specific products and develop misincorporation. The PCR buffer gives specific pH
to Taq polymerase and often contains Tris-HCL, KCl and sometimes MgCl2. To supply
individual bases for the polymerase enzyme to synthesize new DNA strands dNTPs are added.
The dNTPs consist of dATP, dCTP, dGTP, and dTTP and these act as fundamental units to aid
the formation of the DNA strands as the whole purpose behind PCR is to produce an unlimited
number of double stranded DNA (Thermofisher Scientific, 2016), (Department of
Environmental sciences, 2004).

DNA polymerase can initiate DNA replication by adding nucleotides to primers. Short
fragments of single stranded DNA (15-30 nucleotides) are referred to as PCR primers. They
consist of the forward and reverse primer. The right primer must be used to produce the desired
DNA sequence. Thus, for successful DNA amplification a proper primer design is necessary.
Primers are designed as such they are always specified in the 5' to 3' direction. They should
have a GC content between 40-60% and a C or G at the 3' to promote binding. The last 5 bases
of a primer should contain at least 2G or C bases at its 3' end as G-C have a strong binding than
A-T base pairs. Three or more G or C nucleotides at the 3' should be avoided as it could cause
nonspecific priming. The melting temperature between two primers should not differ by more
than 5℃. Most importantly the primers (Forward and Reverse) need to be designed without
complementarity between the two especially at their 3’ which could result in the rise of primer
dimers by annealing.

18
Primer dimers are shorter in length and can be amplified quickly which is referred to as
nonspecific amplification. Care should be taken during primer designing to avoid self-
complementarity as it can cause in self-priming and direct repeats due to which an imperfect
alignment with the template’s target area is created. Non-specific amplification and mispriming
can possibly be caused due to higher primer concentration and low primer concentrations could
result in low or no amplification of the target of interest (Alfandary, 2015), (Department of life
sciences, 2004).

DNA polymerases play the major role in replicating DNA and Taq polymerase is the best
enzyme for PCR. It is highly thermo stable as it is isolated from the bacterium Thermus
aquaticus which survives in extremely hot environments. Therefore, it could efficiently create
DNA strands under intensive heat conditions of the PCR reaction. The enzyme has a half-life
of approximately 40 minutes at 95℃ and adds nucleotides at a rate of about 60 base pairs per
second at 70℃ and can amplify of about 5kb of length. PCR yields can be improved by
increasing the amount of DNA polymerase but may produce non-specific PCR products
(Department of life sciences, 2004). Taq polymerase is added finally as to prevent
contamination and if it is added before the addition of the DNA template it may polymerize
with the primers.

DNA template added is the target gene you wish to amplify. The concentration of its addition
depends on the source and concentration of the target gene. Higher amounts of DNA input can
increase the yield of nonspecific amplification and lower inputs can result in reduced yields.
The DNA template is added only to the reaction tube and not to the control. It is to confirm the
absence of contamination in the experimental result (Clark and Pazdernik, 2013). The PCR
machines also known as thermal cyclers are instruments used to amplify DNA. They are
equipped with a heated lid to prevent the condensation and evaporation of water from the
reaction samples on the inside of the lid (Smith, 2013).

The PCR is a three step process. The initial step of PCR is denaturation where the tube
containing the sample is heated to 94℃ which causes the double stranded DNA to separate
into two strands by breaking the hydrogen bonds between nucleotides resulting in a single
stranded template. The second phase is known as primer annealing as PCR targets strands that
are bound to primers. Here, two primers are used for each one of the newly synthesized strand.
The primers bind to the 3' end of the DNA template which is the beginning of the sequence to
be copied. In order to facilitate primer annealing the tube is cooled down and binding of primers
occurs between 50-60℃. The optimum temperature for primer annealing was found to be 58℃.
Thereafter, the two single stranded templates are ready to be copied. In the third phase called
extension the temperature of the reaction is raised to 72℃ so that Taq polymerase extends the
primers forming new strands of DNA (Butler, 2012), (Kavya, 2015).

19
Two identical copies from the parental DNA are formed after completing extension. After
creating the two DNA copies the cycle begins again with the replicated DNA. Each replicated
DNA creates two copies of DNA after every cycle and after approximately 30-40 cycles which
generally takes a few hours’ a billion copies of the original DNA have been created (Clark and
Pazdernik, 2013)

Figure 15. PCR amplification (Laura, 2015)

The results of the PCR was visualized using gel electrophoresis. A 100 base pair DNA ladder
was added as the DNA marker to determine the band size of the DNA samples as shown in
figure 14. The band size obtained was 200 base pairs. But an error occurred during the loading
of samples into the wells. Usually a gap is left between each sample to avoid overlapping of
the samples but in the procedure done above a gap was not left between the ladder, Group 1A
and its control which should be prevented in further experiments.
PCR has been useful in human identification, detecting viral diseases. Scientists have also
looked for specific traits in plant and animal breeding using PCR. This technique is used in
many research labs including applications in forensics, diagnostics and genetic testing
(Garibyan and Avashia, 2013)

To conclude with, the intensity concentration obtained in the group 1A sample was 100 ng/µl
and group 1B was found to be 720 ng/µl using a spectrophotometer. The size of both DNA
fragments approximately consisted of 200 base pairs.

20
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24
8. Appendix
In order to find the

 Mass of Agarose powder used in preparation of

0.8% Agarose gel 0.8/100 x 50ml

= 0.4g

1% Agarose gel 1/100 x 50ml

= 0.5g

 Final concentrations of the of the PCR components as given in table 2, the following
calculations were done.

1. MgCl2

C1V1=C2V2
25mM x 1.5µl = C2 x 25µl
therefore, C2 = (25mM x 1.5µl)/25 µl

C2 = 1.5mM

2. PCR buffer

C1V1=C2V2
5X x 5µl = C2 x 25µl
therefore, C2 = (5X x 5µl)/25 µl

C2 = 1X

3. dNTP

C1V1=C2V2
10mM x 0.5µl = C2 x 25µl
therefore, C2 = (10mM x 0.5µl)/25 µl

C2 = 0.2mM

4. Forward primer

C1V1=C2V2
2µM x 2.5µl = C2 x 25µl
therefore, C2 = (2µM x 2.5µl)/25 µl

C2 = 0.2µM

25
5. Reverse primer

C1V1=C2V2
2µM x 2.5µl = C2 x 25µl
therefore, C2 = (2µM x 2.5µl)/25 µl

C2 = 0.2µM

4. Taq

C1V1=C2V2
5U/µl x 0.25µl = C2 x 25µl
therefore, C2 = (5U/µl x 0.25µl)/25 µl

C2 = 0.05U/µl

 Volume of water used in

Reaction tube = 25µl- 13.25µl

=11.75µl

Control = 25µl- 12.25µl

=12.75µl

26