DNA Extraction

By
Dr.Hanaa Mahmoud Mohamed
Prof. Genetic and Molecular Biology
DNA extraction: is a process of purification of DNA from sample using a
combination of physical and chemical methods.
oBreaking the cells open commonly referred to as cell disruption or cell lysis,
to expose the DNA within.
oRemoving membrane lipids by adding a detergent or surfactants which also
serves in cell lysis.
oRemoving proteins by adding a protease.
oRemoving RNA by adding an Rnase.
oDNA purification from detergents, proteins, salts and reagents used during
cell lysis step.
The most commonly used procedure is ethanol precipitation usually by ice-cold
ethanol or isopropanol. Since DNA is insoluble in these alcohols, it will
aggregate together, giving a pellet upon centrifugation.
Refinements of the technique include adding a chelating agent to sequester
divalent cations, such as Mg2+ and Ca2+, which prevents enzymes like DNase
from degrading the DNA. After isolation, the DNA is dissolved in slightly alkaline
buffer, usually in the TE buffer, or in ultra-pure water.
 Overview of DNA Extraction

Break down Centrifuge to Precipitate

the cell separate the the DNA
membranes solids from using
the dissolved isopropanol
DNA

Centrifuge to
separate the
DNA from
the dissolved
salts and
Dissolve Wash the sugars
DNA DNA pellet
with Ethanol
and dry the
pellet
Spin column-based nucleic acid purification is a solid phase extraction
method to quickly purify nucleic acids.
This method relies on the fact that nucleic acid will bind to the solid phase
of silica under certain conditions.
 The stages of the method
• Lysis: The cells of a sample are broken open with a lysis procedure.
• Bind: A buffer solution is then added to the sample along with ethanol or
isopropanol. This forms the binding solution.
The binding solution is transferred to a spin column and the column is put in a
centrifuge.
The centrifuge forces the binding solution through a silica gel membrane that
is inside the spin column. If the pH and salt concentration of the binding
solution are optimal, the nucleic acid will bind to the membrane as the
solution passes though.
• Wash: The flow-through is removed and a wash buffer is added to the column.
The column is put in a centrifuge again, forcing the buffer through the membrane.
This washes any remaining impurities from the membrane, leaving only
the nucleic acid bound to the silica gel.
• Elute: The wash buffer is removed and an elution buffer (or simply water) is
added to the column. The column is put in a centrifuge again, forcing the buffer
through the membrane. The elution buffer removes the nucleic acid from the
membrane and it is collected from the bottom of the column.
• A diphenylamine (DPA) indicator will confirm the presence of DNA.
• to produce a blue-colored compound.
• DNA concentration can be determined by measuring the intensity of
absorbance of the solution at the 600 nm with a spectrophotometer and
comparing to a standard curve of known DNA concentrations.
• Measuring the intensity of absorbance of the DNA solution at wavelengths
260 nm and 280 nm is used as a measure of DNA purity.
• DNA absorbs UV light at 260 and 280 nm, and aromatic proteins absorb UV
light at 280 nm; a pure sample of DNA has a ratio of 1.8 at 260/280 and is
relatively free from protein contamination.
• A DNA preparation that is contaminated with protein will have a 260/280 ratio
lower than 1.8.
 Electrophoresis of DNA

DNA can be quantified by cutting the DNA with a restriction enzyme, running
it on an agarose gel, staining with ethidium bromide or a different stain and
comparing the intensity of the DNA with a DNA marker of known
concentration
• The polymerase chain reaction (PCR) is a biochemical technology in molecular
biology used to amplify a single copy or few copies of a piece of DNA across
several orders of magnitude, generating thousands to millions of copies of a
particular DNA sequence.
• PCR is now a common and often indispensable technique used in
medical and biological research labs for a variety of applications.
These include DNA cloning for sequencing, DNA-based phylogeny, or
functional analysis of genes; the diagnosis of hereditary diseases; the
identification of genetic fingerprints (used in forensic sciences and
paternity testing); and the detection and diagnosis of infectious
diseases.
• The method based on thermal cycling, consisting of cycles of repeated
heating and cooling of the reaction for DNA melting and enzymatic
replication of the DNA.
• In the first step, the two strands of the DNA double helix are physically
separated at a high temperature in a process called DNA melting.
• In the second step, the temperature is lowered and the two DNA strands
become templates for DNA polymerase to selectively amplify the target
DNA.
• The selectivity of PCR results from the use of primers that are
complementary to the DNA region targeted for amplification under specific
thermal cycling conditions.
Polymerase Chain Reaction
(PCR)
By
Dr. Hanaa Mahmoud Mohamed
Polymerase Chain Reaction
(PCR)
• PCR is a means to amplify a particular piece of DNA
• Amplify= making numerous copies of a segment of DNA
• PCR can make billions of copies of a target sequence of DNA in a few hours
• PCR was invented in the 1984 as a way to make numerous copies of DNA
fragments in the laboratory
• Its applications are vast and PCR is now an integral part of Molecular Biology
 PCR: the in vitro version of DNA Replication
The following components are needed to perform PCR
in the laboratory:
DNA (your DNA of interest that contains the target sequence (1
you wish to copy)
A heat-stable DNA Polymerase (like Taq Polymerase) (2
All four nucleotide triphosphates (3
Buffers (4
Two short, single-stranded DNA molecules that serve as primers (5
Thin walled tubes (6
Thermal cycler (a device that can change temperatures (7
dramatically in a very short period of time)
 PCR

The DNA, DNA

polymerase, buffer,
nucleoside triphosphates,
and primers are placed in
a thin-walled tube and
then these tubes are
placed in the PCR
thermal cycler

PCR Thermocycler
The three main steps of PCR
• The basis of PCR is temperature changes and the effect that these temperature changes have on the DNA.
• In a PCR reaction, the following series of steps is repeated 20-40 times
(note: 25 cycles usually takes about 2 hours and amplifies the DNA fragment of interest 100,000 fold)
Step 1: Denature DNA
At 95C, the DNA is denatured (i.e. the two strands are separated)

Step 2: Primers Anneal

At 40C- 65C, the primers anneal (or bind to) their complementary sequences on the single strands of DNA

Step 3: DNA polymerase Extends the DNA chain

At 72C, DNA Polymerase extends the DNA chain by adding nucleotides to the 3’ ends of the primers.
Heat-stable DNA Polymerase
• Given that PCR involves very high temperatures, it is imperative that a
heat-stable DNA polymerase be used in the reaction.
• Most DNA polymerases would denature (and thus not function properly) at the high
temperatures of PCR.
• Taq DNA polymerase was purified from the hot springs bacterium
Thermus aquaticus in 1976
• Taq has maximal enzymatic activity at 75 C to 80 C, and
substantially reduced activities at lower temperatures.
 Denaturation of DNA

This occurs at 95 ºC mimicking the function of

helicase in the cell.
 Step 2 Annealing or Primers Binding

Reverse Primer

Forward Primer

Primers bind to the complimentary sequence on the

target DNA. Primers are chosen such that one is
complimentary to the one strand at one end of the
target sequence and that the other is complimentary
to the other strand at the other end of the target
sequence.
 Step 3 Extension or Primer Extension

extension

extension

DNA polymerase catalyzes the extension of the

strand in the 5-3 direction, starting at the
primers, attaching the appropriate nucleotide
(A-T, C-G)
The next cycle will begin by denaturing the new •
DNA strands formed in the previous cycle
 The Size of the DNA Fragment Produced in PCR is
Dependent on the Primers
The PCR reaction will amplify the DNA section between the two primers. •
If the DNA sequence is known, primers can be developed to amplify any piece of an organism’s •
DNA.

Forward primer

Reverse primer

Size of fragment that is amplified

The DNA of interest is amplified by a power of 2
for each PCR cycle
For example, if you subject your DNA of interest to 5 cycles of PCR, you
will end up with 25 (or 64) copies of DNA.
Similarly, if you subject your DNA of interest to 40 cycles of PCR,
you will end up with 240 copies of DNA
PCR has become a very powerful tool in molecular biology
• One can start with a single sperm cell or stand of hair and amplify the
DNA sufficiently to allow for DNA analysis and a distinctive band on an
agarose gel.
• One can amplify fragments of interest in an organism’s DNA by choosing
the right primers.
• One can use the selectivity of the primers to identify the likelihood of an
individual carrying a particular allele of a gene.