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Real-Time PCR For Plants

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Theda Lukito

on 24 June 2011

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Transcript of Real-Time PCR For Plants

RT-PCR for Plants PCR itself... What is PCR? an in vitro technique for DNA/RNA amplification from an organism. What do you need for PCR? 2 oligonucleotide primers Polymerase enzyme Template dNTP Thermocycler PCR Process: Denaturation Annealing Elongation PCR Application & Variation Application: DNA Amplification
Disease Detection
DNA Isolation Variation: Nested PCR
Multiplex PCR
Reverse Transkriptase PCR Real Time PCR Disadvantages of PCR? Qualitative only :( Solution: Quantitave PCR! Ex: Real-Time PCR :) Cannot count the amplicons Amplification + counting the amplicons Real Time PCR Advantages of RealTime PCR Accurate High Sensitivity More Samples Less effort No need for post PCR sample handling. Disadvantages of Real Time PCR Expensive! Important Elements: [Mg] & Probe Reaction Condition (Time & Temp.) Primer Sequence How pure is the sample Application Detect infectious agent Gene transcription (quantitative) Detect cancer, gene abnormality Mutation mRNA expression analysis Detect GMO Plants and many more. Methods of Real Time PCR Classic TaqMan System Construction of labelled oligonucleotide probes (TaqMan probe) will emit fluorescence signal when broken down Taq DNA Polymerase 5' -> 3' exonuclease activity degrade fluorescence labelled probe probe labelled by: fluorochrome reporter on one side and fluorochrome quencher on the other side in it's free or fixed form no fluorescent signal emitted why? because the signal emitted by reporter will be absorbed by quencher in annealing process probe will be degraded reporter and quencher separated emission of fluorescence signal amount of signal emitted will be measured and has direct correlation to PCR product RT-PCR with DNA intercalating dyes use of small particled DNA dye dye will slip inside the minor groove of dsDNA this will increase the intensity of the fluorescent signal e.g ethidium bromide SYBR green advantages: compatible with all kinds of primers

cheaper than TaqMan system disadvantages: unspecific coloring

need analysis curve to differentiate specific target RT-PCR with probe hybridization using 2 juztaposed sequence specific probes also known as HybProbes each probe has: 1 fluorescence reporter
1 fluorophore donor at 3'
1 fluorophore acceptor at 5' both of the probe's sequences are designed to attach to target
sequence near each other in it's free state, probe won't emit fluorescence signal in annealing phase, 2 fluorophore will be near to each other fluorochrome donor will emit light and light will be captured by fluorochrome acceptor this is called resonance energy transfer the amount of fluorescence signal emitted can be
measured at annealing phase and is proportional to target DNA produced RT-PCR with molecular beacons probe with hairpin loop structure labelled with reporter and quencher on each side when probe attaches to complementary target sequence probe structure will be linear this will increase the fluorescence emission this method is often used for identification
of point mutation RT-PCR with scorpions fusing probe and fluorescence molecule together primer and probe are complementary to each other hairpin loop structure the end of hairpin loop is separated from primer to prevent amplification no fluorescence signal will be emitted in hairpin loop structure in annealing phase, the structure will shift to linear emission of fluorescence signal this technique will produce stronger signals let's get in the details of each method Data Analysis 3 cycle of PCR reaction: exponential phase
linear phase
plateau phase exponential phase significant production rate linear phase linear production rate plateau phase production stopped quantification are done automatically by computer signal emission will be calculated to form a graphic quantification can be done by: absolute quantification -> determine the amount of product's copy
relative quantification -> physiology and pathology studies absolute quantification uses calibration curve based on the amount of product's copy Math model used efficiency calibrated model Ct model both model uses the same experimental system Ct is the first curve to
cDNA input graph will be calculated to determine efficiency of amplification nevertheless, efficiency calibrated model are more widely used So... This is the end of our presentation. Hope you have understand more about Real Time PCR! Thank you Danke Gratzie Arigatou Xie Xie Kamsa Hamnida Terima Kasih PCR Target Sequence & Composition The instrument : CCD camera & computer software.
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