Genomic DNA purification
IMBB 2013
Why purify DNA?
The purpose of DNA purification from the cell/tissue is to
ensure it performs well in subsequent downstream
applications, e.g. Polymerase Chain Reaction (PCR),
microsatellite analysis etc.
Ideally, the DNA should be free of contamination with
• Protein
• Carbohydrate
• Lipids
• Other nucleic acid (i.e. DNA free of RNA)
• Tannins, phenolics
Genomic DNA extraction from animal tissue
Silica spin column purification of DNA
Prepare
lysate using
Digestion
Buffer
Apply lysate
to column
and spin
Apply wash
buffer 1 to
column and
spin
Apply wash
buffer 2 to
column and
spin
Elute DNA
with low
salt buffer
Genomic DNA extraction from animal tissue
Chelex Method
Add tissue sample to 20%
(w/v) Chelex in Water
Heat 95oC for 5 min
Centrifuge
Remove supernatant
Genomic DNA extraction from plant leaves:
Modified Dellaporta method
Lysis in SDS-DTT extraction buffer
Precipitate proteins
Breaks open cells
and releases DNA
Chloroform extraction
RNase treatment
Forms complexes with lipids
and proteins, causing them to
precipitate out of solution
Digests RNA
Chloroform extraction
Isopropanol precipitation
Ethanol precipitation
Dry DNA pellet
Redissolve
Purifies and
concentrates the DNA
Polymerase chain
reaction
What is PCR?
•
•
The polymerase chain reaction (PCR) is a relatively simple technique developed in
early 1980’s to make many copies of sequence-specific DNA fragments in vitro.
Also called DNA amplification.
•
PCR is one of the most useful techniques in biosciences labs today due to its speed
and sensitivity.
– Traditional techniques to amplify DNA require days or weeks; PCR can be
performed in as little as 2-3 hours.
– Many molecular analyses require the input of significant amounts of biological
material; PCR requires as little as one DNA molecule.
•
These features make PCR extremely useful in basic research and commercial
applications:
– DNA (and RNA) cloning
– DNA (and RNA) detection (e.g. diagnostics)
– DNA (and RNA) quantitation
– Genotyping
– DNA-based identification (DNA Barcoding)
What is PCR?
• The polymerase chain reaction (PCR) is a relatively simple in vitro
technique to amplify (make multiple copies of) a specific sequence
(i.e. a small region or fragment) of DNA from a complex mixture of
DNA.
DNA from sample
Target DNA (template)
What is PCR?
• The polymerase chain reaction (PCR) is a relatively simple in vitro
technique to amplify (make multiple copies of) a specific sequence
(i.e. a small region or fragment) of DNA from a complex mixture of
DNA.
DNA from sample
Target DNA (template)
How does PCR work?
The method involves using a pair of short DNA sequences called
primers, or oligonucleotides, which are made in the laboratory.
The primers are designed to be complimentary to the segment of
the DNA to be amplified.
The reaction
A sample of target DNA is mixed with
• the primers
• 4 nucleotides (dNTPs) (the building blocks of DNA),
• a DNA polymerase (DNA replication enzyme which synthesises
new copies of DNA)
• Reaction buffer
PCR Basics
Step 1
The reaction is heated to about 95oC to denature the DNA
(strand separation). This is called ‘denaturation’.
PCR Basics
Step 1
The reaction is heated to about 95oC to denature the DNA
(strand separation). This is called ‘denaturation’.
PCR Basics
Step 2
By reducing the reaction temperature to about 45-65oC, the
primers in the reaction specifically bind (‘anneal’) to
complementary regions on the target DNA.
This is called ‘primer annealing’ or ‘annealing’.
PCR Basics
Step 3
The reaction temperature is then raised to 72oC.
At this temperature the DNA polymerase make two new strands
of the target DNA, beginning at where the primers have
bound. This step is known as ‘extension’ or ‘elongation’
because the polymerase extends or elongates the primer,
using the complementary strand as a template.
To withstand the high temperature of the PCR, a thermostable
DNA polymerase is used (e.g. Taq DNA pol).
PCR Basics
The three steps, or ‘cycle’, is repeated 30-35 times.
As PCR progresses, the DNA generated is itself used as a
template for replication, setting in motion a chain reaction
in which the DNA template is exponentially amplified.
(The amount of target DNA is doubled with each cycle.)
Buffer with
magnesium
A PCR includes
Reaction
tube
DNA from sample
Target DNA (template)
Taq DNA
polymerase
Primer 1
Primer 2
Deoxyonucleotide
triphosphates
(dNTPs)
After mixing these components, the reaction tube is placed
into a thermocycler,
which takes the reaction through a series of three different
temperature steps for varying short amounts of time (30-60
sec).
This temperature series is referred to as one “cycle” of
amplification.
Each cycle consists of the following 3 steps:
One PCR cycle
1
3
2
A typical PCR has 30-35 cycles
PCR movie
PCR movie for IMBB_2013.flv
Figure 8-45b Molecular Biology of the Cell (© Garland Science 2008)
Animal
?
Muscle
sample
DNA
CO1 gene PCR
CO1 gene (~1500 bp)
PCR
PCR product (~650-700 bp)
Leaf sample
?
DNA
rbcL gene PCR
rbcL gene (~1430 bp)
PCR
PCR product (~600 bp)
Bioneer AccuPower PCR PreMix is a ready-to-use PCR reagent,
in individual PCR tubes, lyophilised and stable.
Thank you