INTRODUCTION

DNA, or deoxyribonucleic acid, is the hereditary material in humans and almost all other
organisms. Nearly every cell in a persons body has the same DNA. Most DNA is located in the
cell nucleus
The information in DNA is stored as a code made up of four chemical bases: adenine (A),
guanine (G), cytosine (C), and thymine (T). Human DNA consists of about 3 billion bases, and
more than 99 percent of those bases are the same in all people. The order, or sequence, of these
bases determines the information available for building and maintaining an organism, similar to
the way in which letters of the alphabet appear in a certain order to form words and sentences.
DNA bases pair up with each other, A with T and C with G, to form units called base pairs. Each
base is also attached to a sugar molecule and a phosphate molecule. Together, a base, sugar, and
phosphate are called a nucleotide. Nucleotides are arranged in two long strands that form a spiral
called a double helix.
An important property of DNA is that it can replicate, or make copies of itself. Each strand of
DNA in the double helix can serve as a pattern for duplicating the sequence of bases. This is
critical when cells divide because each new cell needs to have an exact copy of the DNA present
in the old cell.
OBJECTIVES
1. To extract DNA from small quantities of buccal cells (cheeks cell)
2. To use PCR to amplify a DNA fragment
3. To examine the PCR products using gel electrophoresis

MATERIALS
5A Extraction of genomic DNA from buccal cells
P 1000 & sterile tips (Blue)
1.5 ml of eppendoft tubes (sterile)
Sterile saline
DNAZol direct solution
Micro-centrifuge
Cotton swab or plastic stirrer

P 20 &sterile tips (Yellow)

5B Polymerase chain reaction
10x buffer
58mM magnesium chloride
Primers
dNTPs stock solutions
Thermostable Taq polymerase

METHODS
5A
1.1000L of sterile saline was pipette into a clean eppendoft tube and labeled
2. The inside of cheeks was scraped gently with cotton swab
3. The swab then was swirled in the saline to remove the cheeks cell
4. The eppendoft tube was spinned and collected on a micro-centrifuge (full speed,30 seconds)
5. The supernatant was removed and cell pellet was washed in 1ml of sterile saline

5B
1. The following content was added in a 0.5ml of PCR tube (except the DNA sample)
Components
Sterile dH2O
10x buffer
MgCl2
dNTPs
Primer( Forward)
Primer(Backward)
Taq Polymerase
DNA sample
Total

Concentration(mM)

10x
5
10
10
10
5 U/L

Volume per Reaction

(l)

Final concentration
(mM)

2. The solution is mixed by vortex followed by a brief ( 10 sec) pulse in the centrifuge
3.Once everyone has prepared the PCR mix, add 5 L of crude DNA extract and immediately
place in the thermocycler block
4.Run the following cycling program
Initial Denaturation
Denaturation
Annealing
Extension
Final cycle

Temp
94C
94 C
55 C
72 C
72 C
4 C

5. After completion was stored at -20C

RESULT

Time
10 min
30s
15s
1 min
10 min

Cycle x
1
All 35 cycle

DISCUSSION
The purpose of experiment is to use DNA sample from the cells that line inside of our
mouth and amplify it with polymerase Chain Reaction (PCR).
PCR is known as a laboratory method used for making a very large number of copies of short
sections of DNA called amplifying. It enables specific genes of interest to be detected or
measured.
Human DNA is made up of repeating sequences of four bases adenine, thymine, guanine, and
cytosine.These sequences form two strands that are bound together in a double helix structure by
hydrogen bond. Each strand of the helix is a complement of the other. It is the difference in the
sequence of these bases on each strand of DNA that leads to the uniqueness of each person's
genetic makeup. The arrangement of these bases in each gene is used to produce RNA in
transcription, which in turn produces a protein. The expression of these genes leads to the
production of a large number of proteins that make up our bodies.
In order to amplify the DNA of interest separation of 2 strands of double helix DNA has to be
made. The separation of the buccal cell occurred when the sample was heated to 94C for 10
min. Once the strands separate, the sample was cooled slightly before forward and reverse
primers was added to bind to the single DNA strands of the buccal cell whereby "forward" and
"reverse" in reference to the direction that the bases within the section of DNA are copied. After
the two primers attached to each strand of the DNA, Taq polymerase then copies the DNA
sequence on each strand of the helix, forming two double stranded sections of DNA, each with
one original half and one new half.
In PCR experiment, Taq Polymerase plays important and significant role as it copies DNA to
make new strands and does not break down at very high temperatures. Beside Taq polymerase
and primers, there are others essential components used in PCR, for instance magnesium
chloride, Deoxynucleoside triphosphates(dNTPs) and buffers. Each of this components important
to assist Taq polymerase in annealing of DNA strands thus ensure the PCR process was carried
out successfully .
Magnesium chloride in PCR acts as cofactor and catalyst required by Taq polymerase to create
the DNA chain in replication process. The concentration of Magnesium chloride influences the
productivity and fidelity of polymerases. Therefore it is important to establish an optimal
concentration before PCR experiment was conducted. On the hand, deoxynucleoside
triphosphates(dNTPs) is used by Taq polymerase to extend an annealed primer during extension

of DNA strands as it consists of four nucleotides ATCG. Meanwhile buffers solution (TBE) was
used to provide a suitable chemical environment for optimum activity and stability for Taq
polymerase to function during annealing process in PCR.
The process of denaturing and annealing of DNA strands was repeated once again whereas each
of the two double strands of buccal cell formed before separated to make four single strands and,
when cooled, the primers and polymerase acts by making four double strand sections. Hence
result in the number of DNA strands double in each cycle.Within 30 to 40 cycles, as many as a
billion copies of the original DNA section was produced.
The most common way of seeing the results of PCR is by running a gel electrophoresis. Gel
electrophoresis was used in this experiment to separate DNA molecules of buccal cell according
to their size .DNA molecule is known to be negatively charged, hence enable it to be pulled
through the gel by an electric field. Theoretically small DNA molecules move more quickly
through the agarose gel than larger DNA molecules. This thus produce a series of bands, with
each band containing DNA molecules of a particular size whereby the bands furthest from the
start of the gel are known to contain the smallest fragments of DNA and the bands closest to the
start of the gel are known to contain the largest DNA fragments.
Based on result in gel electrophoresis, the fragments of DNA of buccal cell(cheeks cell) cannot
be seen under the UV light when being photographed. This is because there were many errors
encountered during PCR experiment and gel electrophoresis . For instance, degraded DNA
template during PCR amplication. The degradation may be caused by contamination of the
template. The causatives of contamination are the nucleases from hands that are transferred to the
template while undertaking the process. This could be avoided by wearing gloves on both hands
before the DNA extraction procedure.It is also important to have two controls in PCR experiment
which usually sufficient to eliminate most potential confounding variables.
Other than that, the reannealing temperature used could be the source of error in PCR
experiment. This is because higher temperature than the required condition will results the
disruption of annealing process thereby producing an unanneal DNA. As a consequence,
extension process is inhibited thereby producing no products or very low yield. In contrast, lower
temperature gives false bands due to non-specific amplifications. In gel electrophoresis, there is
a chance where the buffer or agarose used are expired and lose its activeness to separate the
DNA fragments and visualize DNA sample. Other than that, the Amount of voltage applied
maybe too low hence result in failure of DNA fragments to move across electric field during
electrophoresis process

CONCLUSION
Based on result obtained, it can be said the main of objectives of this experiment is achieved
whereas the DNA of interest was extracted successful. This experiment helps to elucidate the
DNA replication process and how it assists DNA profiling (DNA typing, genetic fingerprinting,
DNA testing).It also has the ability to diagnose certain diseases and very efficient in the sense
that it only takes a few hours to get a result using any desirable DNA sample.

REFERENCES
1. White, Bruce A., ed. PCR Cloning Protocols: From Molecular Cloning to Genetic
Engineering. Methods in Molecular Biology, Volume 67. Totowa: Humana Press Inc 1997.
2. Berg, J.M., Stryer, L., and Tymoczko, J.L. Biochemistry 5th ed. New York: W. H. Freeman and
Company, 2002.
3. http://www.shmoop.com/dna/pcr-polymerase-chain-reaction.html
4. http://molecular.roche.com/pcr/Pages/Process.aspx.html