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

Add tissue sample to 20% (w/v) Chelex in Water 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 Chloroform extraction Breaks open cells and releases DNA RNase treatment Digests RNA Forms complexes with lipids and proteins, causing them to precipitate out of solution Chloroform extraction Isopropanol precipitation Purifies and concentrates the DNA Ethanol precipitation Dry DNA pellet Redissolve

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) Prior to the development of PCR, the most common techniques to amplify DNA was subcloning, which uses enzymes to cut and paste DNA fragments together. The DNA sequence of interest is cut away from the surrounding DNA, then ligated to a specialized DNA molecule (a vector) with the necessary signals for DNA replication in bacteria. The vector DNA, which now includes the DNA of interest, is introduced into a bacterial cell, and the DNA is replicated as the bacterial cell grows and divides. This techniques is very time-consuming and labor-intensive. In addition, the entire vector DNA molecule, not just the DNA sequence of interest, is replicated. In some cases the vector sequences can interfere with the scientific study. The original scientific paper describing PCR was published in Science (Saiki, R.K. et al. (1985) Science 230, 1350–4). The authors used PCR for prenatal detection of a ß-globin gene mutation that causes sickle cell anemia. The ß-globin gene was amplified from fetal DNA obtained from chorionic villus sampling during amniocentesis. This PCR-based method was much faster and at least two orders of magnitude more sensitive than existing methods of detecting the mutation.

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

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

Step 2 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 3 PCR Basics 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.)

A PCR includes Buffer with magnesium Reaction tube DNA from sample Target DNA (template) Taq DNA polymerase Primer 1 Deoxyonucleotide triphosphates (dNTPs) Primer 2

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:

A typical PCR has 30-35 cycles 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