PCR (polymerase chain reaction) is a method to analyze a short sequence of DNA (or RNA) even in samples containing only minute quantities of DNA or RNA. PCR is used to reproduce (amplify) selected sections of DNA or RNA.

1. BABCOCK UNIVERSITY
COLLEGE OF HEALTH AND MEDICAL SCIENCES
BENJAMIN S. CARSON (SNR.) SCHOOL OF MEDICINE
DEPARTMENT OF BIOCHEMISTRY
COURSE : BIOINFORMATICS (MBBT 405)
TOPIC: Polymerase Chain Reaction
BY:
Kolawole, Kayode Daniel (11/3269)
3rd November, 2014

2. Definition
Polymerase chain reaction, (PCR), a technique used to make numerous copies of a specific
segment of DNA quickly and accurately. The polymerase chain reaction enables investigators to
obtain the large quantities of DNA that are required for various experiments and procedures
in molecular biology, forensic analysis, evolutionary biology, and medical diagnostics (Gloria et
al., 2014)
PCR (Polymerase Chain Reaction) is a revolutionary method developed by Kary Mullis in the
1980s. Because DNA polymerase can add a nucleotide only onto a preexisting 3'-OH group, it
needs a primer to which it can add the first nucleotide. This requirement makes it possible to
delineate a specific region of template sequence that the researcher wants to amplify. At the end
of the PCR reaction, the specific sequence will be accumulated in billions of copies (amplicons).
DNA synthesis at one primer is directed toward the other, resulting in replication of the desired
intervening sequence. Also needed are free nucleotides used to build the new DNA strands and a
DNA polymerase, an enzyme that does the building by sequentially adding on free nucleotides
according to the instructions of the template.
Components of PCR
DNA template
The sample DNA that contains the target sequence. At the beginning of the reaction, high
temperature is applied to the original double-stranded DNA molecule to separate the strands
from each other.

3. DNA polymerase
A type of enzyme that synthesizes new strands of DNA complementary to the target sequence.
The first and most commonly used of these enzymes is Taq DNA polymerase (from Thermis
aquaticus), whereas Pfu DNA polymerase (from Pyrococcus furiosus) is used widely because of
its higher fidelity when copying DNA. Although these enzymes are subtly different, they both
have two capabilities that make them suitable for PCR: 1) they can generate new strands of DNA
using a DNA template and primers, and 2) they are heat resistant.
Primers
Short pieces of single-stranded DNA that is complementary to the target sequence. The
polymerase begins synthesizing new DNA from the end of the primer.
Nucleotides (dNTPs or deoxynucleotide triphosphates)
Single units of the bases A, T, G, and C, which are essentially "building blocks" for new DNA
strands.
RT-PCR
(Reverse Transcription PCR) is PCR preceded with conversion of sample RNA into cDNA with
enzyme reverse transcriptase.
How PCR works
PCR is a three-step process that is carried out in repeated cycles;
The initial step is the denaturation, or separation, of the two strands of the DNA molecule. This
is accomplished by heating the starting material to temperatures of about 95° C (203° F). Each
strand is a template on which a new strand is built.
In the second step the temperature is reduced to about 55° C (131° F) so that the primers can
anneal to the template.

4. In the third step the temperature is raised to about 72° C (162° F), and the DNA polymerase
begins adding nucleotides onto the ends of the annealed primers.
At the end of the cycle, which lasts about five minutes, the temperature is raised and the process
begins again. The number of copies doubles after each cycle. Usually 25 to 30 cycles produce a
sufficient amount of DNA.
In the original PCR procedure, one problem was that the DNA polymerase had to be replenished
after every cycle because it is not stable at the high temperatures needed for denaturation. This
problem was solved in 1987 with the discovery of a heat-stable DNA polymerase called Taq, an
enzyme isolated from the thermophilic bacterium Thermus aquaticus, which inhabits hot
springs. Taq polymerase also led to the invention of the PCR machine.
Because DNA from a wide range of sources can be amplified, the technique has been applied to
many fields. PCR is used to diagnose genetic disease and to detect low levels of viral infection.
In forensic medicine it is used to analyze minute traces of blood and other tissues in order to
identify the donor by his genetic “fingerprint.” The technique has also been used to amplify
DNA fragments found in preserved tissues, such as those of a 40,000-year-old frozen
woolly mammoth or of a 7,500-year-old human found in a peat bog.

5. Limitations of PCR and RT-PCR
The PCR reaction starts to generate copies of the target sequence exponentially. Only during the
exponential phase of the PCR reaction is it possible to extrapolate back to determine the starting
quantity of the target sequence contained in the sample. Because of inhibitors of the polymerase
reaction found in the sample, reagent limitation, accumulation of pyrophosphate molecules, and
self-annealing of the accumulating product, the PCR reaction eventually ceases to amplify target
sequence at an exponential rate and a "plateau effect" occurs, making the end point quantification

6. of PCR products unreliable. This is the attribute of PCR that makes Real-Time Quantitative RT-PCR
so necessary (NCBI, 2014).

7. References
 Gloria L, Gaurav S, and Surabhi S.
http://www.britanica.com/EBchecked/topic/468736/polymerase-chain-reaction. Retrieved
on 28th october, 2014.
 National Centre for Biotechnology Information, U.S. National Library of Medicine,
Betheoda MD, USA. Http://www.ncbi.nlm.nih.gov/probe/docs/techpcr. Retrieved on 28th
October, 2014.