DNA EXTRACTION AND
AMPLIFICATION BY USING
PCR
SUBMITTED TO : Dr. AMBREEN AHMAD
MS. AQSA TARIQ
SUBMITTED BY: KHADIJA MUSTAFA
ROLL NO: MPHIL-BT19F19
CONTENTS
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What is DNA extraction?
Purpose of DNA extraction
Sources
Basic steps for DNA extraction
Protocol for DNA extracrion
DNA amplification by PCR
Agarose Gel Electrophoresis
Conclusion
References
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WHAT IS DNA EXTRACTION?
Purification of DNA from a
sample.
first isolation by Friedrich
Miescher
Methods used to isolate
DNA are dependent on the
source, age, and size of
the sample.
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Purpose of DNA Extraction
• To obtain DNA in a
relatively purified
form from nucleus of
a cell
• for genetic analysis,
which is used for
scientific, medical, or
forensic purposes.
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Sources
• living or dead organism
• blood, hair, sperm, tissues,
urine,bones,
nails,
epithelial cells etc
• stored samples
• frozen blood or tissues
• exhumed bones or tissues
• ancient human, animal or
plant samples
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Basic steps for DNA Extraction
• Preparation of a cell
extract.
• Purification of DNA from
cell extract.
• Concentration of DNA
samples.
• Measurement of purity of
DNA concentration
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1. Preparation of a cell extract:
cells have to be separated and the cell membranes have
to be disrupted (physically and chemically)
"Extraction buffer"
EDTA (Ethylene Diamine Tetra Acetate
• removes Mg2+ ions that are essential for preserving the
overall structure of the cell membrane.
SDS (Sodium Dodecyl Sulfate)
• aids in disrupting the cell membranes by removing the
lipids of the cell membranes
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continued.....
• removal of insoluble cell debris.
• Cell debris and partially digested organelles etc. can
be pelleted by centrifugation leaving the cell extract as
a reasonably clear supernatant.
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2. Purification of DNA from cell extract
• cell extract contain significant quantities of detergents,
proteins, salts and reagents used during cell lysis.
• The most commonly used procedures are:
i. Ethanol precipitation.
ii. Phenol–chloroform extraction.
iii. Minicolumn purification.
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1. Ethanol precipitation
usually by ice-cold ethanol or isopropanol.
DNA insoluble in these alcohols
aggregates and forms a pellet upon centrifugation.
improved by increasing of ionic strength
usually by adding sodium acetate
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2. Phenol–chloroform extraction
phenol denatures proteins in the sample.
After centrifugation of the sample, denaturated
proteins stay in the organic phase
while aqueous phase containing nucleic acid is
mixed with the chloroform that removes phenol
residues from solution.
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3. Minicolumn purification
• relies on the fact that the nucleic acids may bind
(adsorption) to the solid phase (silica or other)
depending on the pH and the salt concentration
of the buffer.
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3. Concentration of DNA samples
• most frequent method is ethanol precipitation.
• presence of salt and temperature of -20°C or less,
absolute ethanol will efficiently precipitate polymeric
nucleic acids.
• in concentrated solution of DNA glass rod used to pull out
the adhering DNA strands
• in dilute solutions DNA can be collected by centrifugation
and redissolving in an appropriate volume of water.
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4. Measurement of purity of DNA concentration.
• measured by UV absorbance spectrometry.
• The amount of UV radiation absorbed by a solution of
DNA is directly proportional to the amount of DNA
sample.
• Usually absorbance is measured at 260 nm, at this wave
length an absorbance of 1.0 corresponds to 50 µg of
double-stranded DNA per ml.
• With a pure sample of DNA the ratio of the absorbancies
at 260 nm and 280 nm (A260/A280) is1:8.
• Ratios of less than 1:8 means contaminated.
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Nucleic Acid Analysis via UV Spectrophotometry
DNA Absorption Spectra
By measuring the amount of light absorbed by your sample at specific
wavelengths, it is possible to estimate the concentration of DNA and
RNA. Nucleic acids have an absorption peak of 1 OD at ~260nm.
[dsDNA] ≈ A260 x (50 µg/mL)
[ssDNA] ≈ A260 x (33 µg/mL)
[ssRNA] ≈ A260 x (40 µg/mL)
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PCR
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Agarose Gel Electrophoresis
• analysis of nucleic acids and proteins.
• Agarose gel electrophoresis is for the preparation and
analysis of DNA.
• Gel electrophoresis is a procedure that separates
molecules on the basis of their rate of movement through
a gel under the influence of an electrical field.
• We will be using agarose gel electrophoresis to
determine the presence and size of PCR products.
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Electrophoresis Equipment
Power supply
Cover
Gel tank
Electrical leads

Casting tray
Gel combs
Preparation of Agarose gel
An agarose gel is prepared by
combining agarose powder and
a buffer solution.
Buffer
Flask for boiling
Agarose
Melting the Agarose
Agarose is insoluble at room temperature (left).
The agarose solution is boiled until clear (right).
Gently swirl the solution periodically when heating to allow all the grains of agarose
to dissolve.
***Be careful when boiling - the agarose solution may become superheated and
may boil violently if it has been heated too long in a microwave oven.
Preparing the Casting Tray
Seal the edges of
the casting tray
and put in the
combs. Place the
casting tray on a
level surface.
None of the gel
combs should be
touching the
surface of the
casting tray.
Pouring the gel
Allow the agarose solution to cool slightly (~60ºC) and then carefully
pour the melted agarose solution into the casting tray. Avoid air
bubbles.
When cooled, the agarose polymerizes, forming a flexible gel. It should
appear lighter in color when completely cooled (30-45 minutes).
Carefully remove the combs and tape.
Place the gel in the electrophoresis chamber.
DNA
buffer 

wells
Cathode
(negative)



Anode
(positive)
Add enough electrophoresis buffer to cover the gel to a depth of
at least 1 mm. Make sure each well is filled with buffer.
Sample Preparation
Mix the samples of DNA with the 6X sample loading buffer (w/ tracking
dye). This allows the samples to be seen when loading onto the gel, and
increases the density of the samples, causing them to sink into the gel
wells.
6X Loading Buffer: 
 Bromophenol Blue (for color)
 Glycerol (for weight)
Loading the Gel
Carefully place the pipette tip over a well and gently expel the sample.
The sample should sink into the well. Be careful not to puncture the
gel with the pipette tip.
Running the Gel
Place the cover on the electrophoresis chamber, connecting the electrical
leads. Connect the electrical leads to the power supply. Be sure the leads
are attached correctly - DNA migrates toward the anode (red). When the
power is turned on, bubbles should form on the electrodes in the
electrophoresis chamber.
• DNA is negatively charged.
• When placed in an electrical field, DNA will migrate toward the positive
pole (anode).
• An agarose gel is used to slow the movement of DNA and separate by size.
H

O2

DNA
-
+
• Polymerized agarose is porous,
allowing for the movement of DNA
Power
Scanning Electron Micrograph
of Agarose Gel (1×1 µm) 
Cathode
(-)
 wells
 Bromophenol Blue
DNA
(-)

Gel
Anode
(+)
After the current is applied, make sure the Gel is running in the correct
direction. Bromophenol blue will run in the same direction as the DNA.
DNA Ladder Standard
 12,000 bp
 5,000
DNA
migration
Note: bromophenol
blue migrates at
approximately the
same rate as a 300
bp DNA molecule
bromophenol blue
+
 2,000
 1,650
 1,000
 850
 650
 500
 400
 300
 200
 100
Inclusion of a DNA ladder (DNAs of know sizes) on the gel makes it easy to
determine the sizes of unknown DNAs.
Staining the Gel
• Place the gel in the staining tray containing warm diluted stain.
• Allow the gel to stain for 25-30 minutes.
• To remove excess stain, allow the gel to destain in water.
• Replace water several times for efficient destain.
Staining the Gel
• Ethidium bromide binds to DNA and fluoresces under UV light,
allowing the visualization of DNA on a Gel.
• Ethidium bromide can be added to the gel and/or running buffer
before the gel is run or the gel can be stained after it has run.
***CAUTION! Ethidium
bromide is a powerful
mutagen and is moderately
toxic. gloves must be used
Ethidium Bromide requires an ultraviolet light source to visualize
Visualizing the DNA (ethidium bromide)
DNA ladder
 1
2
3
4
5
6
7
DNA ladder
8 
wells
 5,000 bp
 2,000
 1,650
 1,000
 850
 650
 500
 400
 300
 200
 100
PCR Product
Primer dimers
+
-
-
+
-
+
+
-
Safer alternatives to Ethidium Bromide
 Methylene Blue
 BioRAD - Bio-Safe DNA Stain
 Ward’s - QUIKView DNA Stain
 Carolina BLU Stain
…others
advantages
disadvantages
Inexpensive
Less toxic
No UV light required
No hazardous waste disposal
Less sensitive
More DNA needed on gel
Longer staining/destaining time
Conclusion
• DNA islolation is the most critical point in getting a pure
DNA and its copies because if the isolation is poor then
yield of PCR will decrease.
• similarly PCR components purity is much neccessary for
a high yield
• So we can coclude that these processes if done properly
result in production of pure DNA copies that can be further
used for research, in medicines, in forensics etc
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References
• “The Polymerase Chain Reaction”
http://avery.rutgers.edu/WSSP/StudentScholars/project/archives/onions/pcr.html
• “Polymerase Chain reaction” http://www.tulane.edu/~wiser/methods/handouts/pcr.PDF
• Diagrams from : http://allserv.rug.ac.be/~avierstr/principles/pcrani.html
• Purves, Sadava, Orians, Heller. “Life.” 6th ed. Sinauer Associates, 2001.
• “Mechanism of PCR.” http://usitweb.shef.ac.uk/~mba97cmh/tutorial/pcr.htm
• “The polymerase Chain Reaction”www.faseb.org/opar/bloodsupply/pcr.html
• Kainz P. (2000) The PCR plateau phase- towards an understanding of its limitations.
Biochem Biophys Acta, 1494: 23−27.
• Bustin SA. (2004) A to Z of Quantitative PCR. LaJolla, California: International University
Line.
• Chen B-Y, and Janes HW. (2002) PCR Cloning Protocols, Second Edition. Totowa, New
Jersey: Humana Press.
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