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6 Cards in this Set
- Front
- Back
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What is the principle of realtime PCR? |
amplification of target nucleic acid (lag, exponential, saturation phase) detection of amplifed product during each cycle with fluorescent tag results transferred in "real time" (threshold cycle values generated) |
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What steps are involved in the general workflow for real time PCR? |
RNA isolation (cell lysis) [rna]ug/ml= A260 x 40 ug/ml x dilution factor cDNA synthesis (mrna -> cdna) use Reverse Transcriptase, dntps, polymerase RT-PCR (gene target specific probes+primers) master mix cDNA |
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Compare traditional PCR and RT-PCR |
Traditional Gel electrophoresis, not quantitative, short dynamic range (2logs), low resolution, non automated, sized based discrimination only results aren't numbers RT-PCR in real time, can be qantified, no post PCR processing, high throughput (low contamination risk), wider dynamic range (up to 10^10 fold) more specific, sensitive, reproducable |
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Discuss the 2 assay detection methods |
SYBR Green sybr green binds to dsDNA, increase in pcr product = increase in intensity no probe, no dimers MAY GIVE FALSE POS enables cDNA/RNA quantification and allelic discrimination TAQMAN use 5' exonuclease activity of taq polymerase, uses reporter and quencher molecules. Reporter dye mols cleaved from probes in each cycle increase fluorescence intensity (relating to amount of PCR product) detects amplification in real time with probe target binding different probes may be required for specific sequences (some expensive) enables cDNA/RNA quantification and allelic discrimination |
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Describe quantisation |
uses threshold cycle values (Ct) threshold value = cycle when first detection of significant increase in product across a threshold Ct>40 = no specific amplification calibrate with housekeeping gene relative standard curve method -> quantise normalised target = target/endogenous control |
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Describe mutation detection with RT-PCR |
perfect match/mismatch (anchor probe, detection probe) uses melting curve analysis Tm 50% probe dissociates from template ( decrease in flourescence) in presence of mutation detection probes melt at LOWER tmperature than that of a perfect match - some of sequence already dont bond bc mismatch |
