2 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 withProteinCarbohydrateLipidsOther nucleic acid (i.e. DNA free of RNA)Tannins, phenolics
4 Genomic DNA extraction from animal tissue Silica spin column purification of DNAPrepare lysate using Digestion BufferApply lysate to column and spinApply wash buffer 1 to column and spinApply wash buffer 2 to column and spinElute DNA with low salt buffer
5 Add tissue sample to 20% (w/v) Chelex in Water Genomic DNA extraction from animal tissueChelex MethodAdd tissue sample to 20% (w/v) Chelex in WaterHeat 95oC for 5 minCentrifugeRemove supernatant
6 Genomic DNA extraction from plant leaves: Modified Dellaporta method Lysis in SDS-DTT extraction bufferPrecipitate proteinsChloroform extractionBreaks open cells and releases DNARNase treatmentDigests RNAForms complexes with lipids and proteins, causing them to precipitate out of solutionChloroform extractionIsopropanol precipitationPurifies and concentrates the DNAEthanol precipitationDry DNA pelletRedissolve
8 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) cloningDNA (and RNA) detection (e.g. diagnostics)DNA (and RNA) quantitationGenotypingDNA-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.
9 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 sampleTarget DNA (template)
10 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 sampleTarget DNA (template)
11 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 withthe primers4 nucleotides (dNTPs) (the building blocks of DNA),a DNA polymerase (DNA replication enzyme which synthesises new copies of DNA)Reaction buffer
12 Step 1PCR BasicsThe reaction is heated to about 95oC to denature the DNA (strand separation). This is called ‘denaturation’.
13 Step 1PCR BasicsThe reaction is heated to about 95oC to denature the DNA (strand separation). This is called ‘denaturation’.
14 Step 2PCR BasicsBy 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’.
15 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).
16 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.)
17 A PCR includes Buffer with magnesium Reaction tube DNA from sample Target DNA (template)Taq DNA polymerasePrimer 1Deoxyonucleotide triphosphates (dNTPs)Primer 2
18 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:
19 A typical PCR has 30-35 cycles One PCR cycle132A typical PCR has cycles