PCR

1. POLYMERASE CHAIN REACTION (PCR)
PCR is a laboratory ( in vitro) techniques for generating large quantities of a
specified DNA.
The introduction of PCR was one of the greatest recent break through in the
chemical and biologic sciences.

2. PCR was invented in 1983 by American
biochemist Kary Mullis at Cetus
Corporation; Mullis and biochemist Michael
Smith, who had developed other essential
ways of manipulating DNA, were jointly
awarded the Nobel Prize in Chemistry in
1993.

3. PCR is valuable because it is simple, sensitive, and powerful and
amplify a single copy of target DNA into an exponential amount of
nucleic acid product over the course of 25 to 40 reaction cycles.
 PRINCIPLE OF PCR :
 The double stranded DNA of interest is denatured to separate into two
individual strands. Each strand is then allowed to hybridize with a primer
(renaturation). The primer- template duplex is used for DNA synthesis (the
enzyme DNA polymerase). These three steps – DENATURATION ,
RENATURATION (primer annealing ) AND SYNTHESIS (primer extension)
are repeated again and again to generate multiple forms of target DNA.

5. 1)A target DNA(100-35,00 bp in length)
2) Two primers (synthetic oligonucleotides of 17-30 nucleotides length) that are complementary to
regions flanking the target DNA.
3) Four deoxyribulosenucleotides (dATP, dCTP, dGTP, dTTP).
4) A DNA polymerase( from the thermophilic bacterium Thermus aquaticus )that can withstand
at a temperature upto 95 deree Celsius . A buffer for the polymerase (Tris-HCL and a salt, such as
KCL, at a pH of 8.3. )
An instrument called a thermal cycler is also required.

6. The original described method of PCR was labor intensive and used
two separate heat blocks and the Klenow fragments from E.coli as the
DNA polymerase .One heat block was set to 95 degree Celsius and
another at 30 degree Celsius. And the amplification reaction then
proceeded by manually transferring reaction tube from heat block to
heat block for several cycle .New Klenow fragment had to be added
after every cycle.
In 1986 , an instrument called thermal cycler was developed.
.In 1988, a heat stable (thermostable) DNA polymerase from the
thermophilic bacterium Thermus aquaticus was used in PCR.

7.  PCR amplifies DNA in three basic steps
1) Denaturation :
single stranded DNA targets are necessary for PCR assays.
the bond in dsDNA separated at a temperature above 90 degree celsius , so
most PCR protocols denature at 94 °C or 95°C.
The time required for denaturation depends upon PCR assay performed most
commonly 15 to 30 sec is used.

8. 2) Primer annealing :
The goal of this step is to hybridize, or anneal, oligonucleotide primers to the denatured,
single-stranded target DNA strands.
Primer-annealing temperatures usually range from approximately 45°C to 65°C for
about 30 seconds to 2 minutes.
A pair of primers is used in standard PCR, one for each strand of dsDNA.
3)Primer extension :
The purpose of primer extension is to produce the PCR products.
Can be accomplished at 68°C to 72°C.
During this step, the DNA polymerase takes the individual dNTPs and adds them to
the 3′ end of each primer that is annealed to the target DNA strand. This reaction usually is
allowed to proceed for 1 to 2 minutes.
The yield of PCR product is initially low over the first several cycles; however, after
about 20 cycles, the yield is high and generates most of the PCR product, or amplicon.
PCR amplicons can be stored at −20°C or analyzed immediately.

11. 1) agarose gel electrophoresis :
Popular in past .
Electrophoresis separates these molecules based on size, charge, and shape.
RNA and DNA possess a net negative charge in solution because of their phosphate
backbones, so an electric field will force these molecules from a negative to a positive electric
pole.
The most common nucleic acid stain used after separation by agarose gel electrophoresis are
ethidium bromide , SYBR GREEN ( I for DNA and II for RNA)

13. 2)REAL TIME PCR/ KINETIC
PCR/HOMOGENOUS PCR
 The method was developed in the early 1990s by Higuchi and coworkers.
PCR amplicons are assayed as they accumulate during real-time PCR after each cycle.
Major benefit to real-time PCR is that the reactions occur in closed tubes that do not have to be
opened for detection.
Real-time PCR uses a fluorescent reporter dye, often in the form of labeled probes or beacons,
sometimes called a fluorophore.
A thermal cycler that uses a UV light source to excite the reporter, and a camera controlled by a
computer system.
Fluorescent peaks are recorded by the computer system as fluorescence intensity versus PCR cycle
number.
Fluorescence is measured by directly monitoring an increase in fluorescence or indirectly by a
process called fluorescence resonance energy transfer (FRET).