PCR

POLYMERASE CHAIN
REACTION
Adinda Citra D.
Winda Siti N.
Nuerpuas Wulan
Mona Soraya
Nadya Karina
Nurkhamidatus Sintia

WHAT IS PCR?
Polymerase chain reaction, PCR, is an
efficient and cost-effective way to copy or
"amplify" small segments of DNA or RNA.
Using PCR, millions of copies of a section of
DNA are made in just a few hours, yielding
enough DNA required for analysis. This
innovative yet simple method allows clinicians
to diagnose and monitor diseases using a
minimal amount ofsample, such asblood or
tissue.

Though PCR occurs in vitro, or outside of

the body in a laboratory, it is based on the
natural process of DNA replication. In its
simplest form, the reaction occurs when a
DNA sample and a DNA polymerase,
nucleotides, primers and other reagents (manmade chemical compounds) are added to a
sample tube. The reagents facilitate the
reaction needed to copy the DNA code

In addition to detecting diseases in a sample, PCR

enables the monitoring of the amount of a virus
present, or viral load, in a persons body. In
diseases such as hepatitis C or human
immunodeficiency virus (HIV) infections, viral load
is a good indication of how sick a person may be
or how well a persons medicine and treatment is
working. Armed with this information, physicians
may determine when to start treatment and the
persons
response
to
treatment,
making
treatment personalized to each individual.

There are three clear steps in each PCR

cycle, and each cycle approximately doubles
the amount of target DNA. This is an
exponential reaction so more than one billion
copies of the original or target DNA are
generated in 30 to 40 PCR cycles.
1. Denaturation
2. Annealing
3. Extension

Denaturation
During the first step of PCR, called
denaturation, the tube containing the sample
DNA is heated to more than 90 degrees
Celsius (194 degrees Fahrenheit), which
separates the double-stranded DNA into two
separate strands. The high temperature
breaks the relatively weak bonds between the
nucleotides that form the DNA code

Annealing
PCR does not copy the all of the DNA in the sample. It copies
only a very specific sequence of genetic code, targeted by the
PCR primers. For example, Chlamydia has a unique pattern of
nucleotides specific to the bacteria. The PCR will copy only the
specific DNA sequences that are present in Chlamydia and
absent from other bacterial species. To do this, PCR uses
primers, man-made oligonucleotides (short pieces of synthetic
DNA) that bind, or anneal, only to sequences on either side of
the target DNA region.
Two primers are used in step two - one for each of the newly
separated single DNA strands. The primers bind to the
beginning of the sequence that will be copied, marking off the
sequence for step three. During step two, the tube is cooled
and primerbinding occurs between 40 and 60 degrees Celsius
(104 140 degrees Fahrenheit).
Step two yields two separate strands of DNA, with sequences
marked off by primers. The two strands are ready to be copied.

Extension
In the third phase of the reaction, called
extension, the temperature is increased to
approximately 72 degrees Celsius (161.5
degrees Fahrenheit). Beginning at the regions
marked by the primers, nucleotides in the
solution are added to the annealed primers by
theDNA polymerase to create a new strand of
DNA complementary to each of the single
template strands.
After completing the extension, two
identical copies of the original DNA have been
made.

After making two copies

of the DNAthrough PCR,
the cycle begins again,
this time using the new
duplicated DNA. Each
duplicate creates two
new copies and after
approximately 30 or 40
PCR cycles, more than
one billion copies of the
original DNA segment
have
been
made.
Because
the
PCR
process is automated, it
can be completed in just
a few hours.