IMBB 2013

Genomic DNA purification

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

Prepare lysate using

Digestion Buffer

Apply wash buffer 1 to

column and spin

Apply lysate to column and spin

Apply wash buffer 2 to

column and spin

Elute DNA with low

salt buffer

Silica spin column purification of DNA

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

RNase treatment

Precipitate proteins

Isopropanol precipitation

Ethanol precipitation

Dry DNA pellet

Redissolve

Lysis in SDS-DTT extraction buffer

Chloroform extraction

Chloroform extraction

Digests RNA

Purifies and concentrates the DNA

Breaks open cells and releases DNA

Forms complexes with lipids and proteins, causing them to

precipitate out of solution

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)

• 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.

What is PCR?

DNA from sample

Target DNA (template)

• 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?

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 reactionA 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

The reaction is heated to about 95oC to denature the DNA (strand separation). This is called ‘denaturation’.

Step 1

PCR Basics

The reaction is heated to about 95oC to denature the DNA (strand separation). This is called ‘denaturation’.

Step 1

PCR Basics

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’.

Step 2

PCR BasicsThe 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).

Step 3

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.)

PCR Basics

Taq DNA polymerase

Primer 1

Primer 2Deoxyonucleotide triphosphates (dNTPs)

A PCR includes

DNA from sample

Target DNA (template)

Buffer with magnesium

Reaction tube

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

2

3

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)

Muscle sample

Animal

DNA

CO1 gene PCR

PCR product (~650-700 bp)

PCR

CO1 gene (~1500 bp)

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rbcL gene PCR

Leaf sampleDNA

PCR

PCR product (~600 bp)

rbcL gene (~1430 bp)

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Bioneer AccuPower PCR PreMix is a ready-to-use PCR reagent, in individual PCR tubes, lyophilised and stable.

Thank you