Polymerase Chain Reaction -WPS Office

Polymerase Chain Reaction.
Polymerase Chain Reaction or PCR- is a technique use to make multiple copies of DNA segment.
The polymerase chain reaction (PCR) was originally developed in 1983 by the American biochemist Kary
Mullis.
Polymerase chain reaction, or PCR, is a laboratory technique used to make multiple copies of a segment
of DNA. PCR is very precise and can be used to amplify, or copy, a specific DNA target from a mixture of
DNA molecules.
Procedures and step
How does PCR work?
The principles behind every PCR, whatever the sample of DNA, are the same.
Five core ‘ingredients’ are required to set up a PCR. We will explain exactly what each of these do as we
go along. These are:
the DNA template to be copied
primers, short stretches of DNA that initiate the PCR reaction, designed to bind to either side of the
section of DNA you want to copy
DNA nucleotide bases? (also known as dNTPs). DNA bases (A, C, G and T) are the building blocks of DNA
and are needed to construct the new strand of DNA.
A- Adenine
C- Cytosine
G- Guanine
T- Thymine
And Taq polymerase enzyme to add in the new DNA bases
PCR involves a process of heating and cooling called thermal cycling which is carried out by machine.
There are three main stages:
Denaturing – when the double-stranded template DNA is heated to separate it into two single strands.
Annealing – when the temperature is lowered to enable the DNA primers to attach to the template DNA.
Extending – when the temperature is raised and the new strand of DNA is made by the Taq polymerase
enzyme.
What happens at each stage of PCR?
Denaturing stage
Denature/Denaturing- to deprive of natural qualities.
During this stage the cocktail containing the template DNA and all the other core ingredients is heated
to 94-95⁰C.
This results in two single strands of DNA, which will act as templates for the production of the new
strands of DNA.
This usually takes between 15-30 seconds.
Annealing stage
During this stage the reaction is cooled to 50-65⁰C. This enables the primers to attach to a specific
location on the single-stranded template DNA by way of hydrogen bonding (the exact temperature
depends on the melting temperature of the primers you are using).
Annealing – when the temperature is lowered to enable the DNA primers to attach to the template DNA.
Elongation
During this final step, the heat is increased to 72⁰C to enable the new DNA to be made by a special Taq
DNA polymerase enzyme which adds DNA bases.
Taq DNA polymerase is an enzyme taken from the heat-loving bacteria? Thermus aquaticus.
This bacteria normally lives in hot springs so can tolerate temperatures above 80⁰C.
The bacteria's DNA polymerase is very stable at high temperatures, which means it can withstand the
temperatures needed to break the strands of DNA apart in the denaturing stage of PCR.
72⁰C is the optimum temperature for the Taq polymerase to build the complementary strand. It
attaches to the primer and then adds DNA bases to the single strand one-by-one.
The following applications of PCR:
1) The amplification of gene fragments as fast alternative of cloning. 2) The modification of DNA
fragments. ... 4) DNA analysis of arachaeological specimens. 5) The detection of mutations relevant for
inherited diseases, malignant transformation or tissue typing.
Spectrophotometry
Spectrophotometry is a standard and inexpensive technique to measure light absorption or the amount
of chemicals in a solution. It uses a light beam which passes through the sample, and each compound in
the solution absorbs or transmits light over a certain wavelength. The instrument used is called a
spectrophotometer.
The spectrophotometry method when applied to biology can measure the amount of radiant energy
contained in a substance and multiple wavelengths of light. ... This is determined by measuring the
wavelength by which peak absorption occurs, which is also known as the absorption maximum.
Spectroscopy-the study of how light interacts with matter.
In molecular biology, quantitation of nucleic acids is commonly performed to determine the average
concentrations of DNA or RNA present in a mixture, as well as their purity.
DNA sequencing
Is the process of working out the order of the building blocks or basis in a strand of DNA.
Before we can sequence the DNA it has to be cut up in smaller pieces that are inserted into plasmid
DNA.
Then put into bacterial cell this makes it possible to produce lots and lots of copies of it as the bacterial
cell multiply. Then isolated from the bacteria for sequencing transferred to a plate then a mixture of
ingredients are added.
*Free DNA bases
" DNA polymerase
*DNA primer
* Modified DNA bases labeled with colored fluorescent flags called Terminator base
To start the sequencing reaction everything is heated to 96 degree celsius these separates the DNA into
two single stand then the temperature is lowered down to 50°Cthis enables the DNA primers to bind to
the plasmid DNA.
The temperature is the then increase to 60°C this allows the DNA polymerase to bind to the DNA
primers then the DNA polymerase starts making a new strand by adding unlabeled DNA bases to the
target DNA it continues to add DNA bases until a Terminator is added.
These Terminator bases are chemically altered so that no more bases can be added to the new strand so
the DNA polymerase won't stop making DNA until a Terminator is added. So when the Terminator is
added the DNA polymerase will stop making DNA and falls away. Then is heated to 96°C to to separate
the new DNA strand from the original strand.
Then the process of heatiiand cooling is repeated again and again to produce a lot of DNA fragments of
different lengths, the length of each fragment depends when a Terminator base is added.
To read the the sequence of the DNA, the various fragments are separated by length using a process
called Gelectrophoresis. A capillary tube is lowered to each wall of the plate, an electrical charge is
applied this causes the negatively change DNA molecules to move through the capillary. Each capillary
contains a phores gel the shorter the fragment of DNA move through the gel more easily than the longer
fragments as a result the fragments are now arrange from the size from shortest to longest.
As the DNA came to the end of the capillary a laser makes the Terminator light up, the color is detected
by a camera and recorded each bases is labeled with different color.
A- fluorescent Green
C- fluorescent Blue
G- fluorescent Yellow
T- fluorescent Red
The shortest is read first the longest is read last.
The sequencing machine records the color of the Terminator base as series of colored block. Each
colored block represent the labeled Terminator base at the end of each fragments of DNA.
By converting the colors into letters we get our sequence of our piece of DNA.

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Polymerase Chain Reaction.

Procedures and step

How does PCR work?

the DNA template to be copied

And Taq polymerase enzyme to add in the new DNA bases

There are three main stages:

What happens at each stage of PCR?

Denature/Denaturing- to deprive of natural qualities.

This usually takes between 15-30 seconds.

Spectroscopy-the study of how light interacts with matter.

*Free DNA bases

" DNA polymerase

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