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PCR & qPCR
| Question | Answer |
|---|---|
| What is PCR? | In vitro amplification of a DNA template |
| What was discovered that made PCR possible? | Thermus aquaticus/thermophilic bacterium |
| What enables temperature cycling | Thermostability of the 'Taq' polymerase |
| Advantages of PCR? (3) | 1. Low amounts of starting material needed 2. Rapid & multiple samples can be analysed simultaneously 3. Sensitive and CAN be quantitative |
| How does PCR work? | Thermo-cycling based. |
| Around what cycle do we see products on the agarose gel? | cycle 25 |
| What are the steps of PCR? | 1. Denaturation 2. Primer annealing 3. Extension |
| What is Tm? | Temperature at which ds & ss states are at equilibrium |
| What happens during the denaturation step? | At 98°C, dsDNA is denatured to ssDNA |
| What happens during primer annealing? | Temperature is lowered (50-75°C) and forward and reverse primer are annealed to the ssDNA. |
| What happens during extension? | At 72°C, thermostable DNAP do primer extension, fuelled by dNTPs. Taq polymerase adds nucleotides to annealed primer. |
| Which bonds are more annoying to do PCR with? | G-C bonds are slightly more stable (3 H bonds) so are more difficult to break - can cause secondary structure problems. |
| In what direction if DNA synthesised? | 5'-3' direction |
| What is a primer? | Small ssDNA molecules that are commercially synthesised |
| Primer design? (2) | 1. Must anneal to the template DNA with high efficiency 2. Must avoid creating Primer-dimers (self-complementary) |
| How is temperature decided for PCR? | 5°C lower than calculated primer Tm |
| what is qPCR? | Quantitative PCR ('real-time' PCR) |
| What is RT-PCR? | Reverse Transcriptase PCR |
| What is RT-qPCR? | Reverse Transcriptase Quantitative PCR |
| What is the Ct value? | The threshold cycle |
| What does qPCR do? | Measures amount of amplified DNA product by monitoring fluorescence emitted during each cycle instead of at a fixed end-point |
| What is an amplicon? | The PCR product |
| Which phase of qPCR is focused on and why? | Exponential phase - as it is the only part linked to the amount of template put in |
| What is the Ct cycle? | Cycle at which amount of PCR product crosses the detection threshold |
| Ways of using fluorescence during qPCR? (2) | 1. Intercalating dyes 2. Sequence specific probes |
| Advantages/Disadvantages of intercalating dyes for qPCR? | cheap but not as accurate |
| How do intercalating dyes work | The dye forms a complex with dsDNA that emits fluorescence when irradiated with its wavelength of light |
| Intercalating dye example please!!! | SYBR Green SYBR-dsDNA absorbs 497nm, emits 520nm |
| How do sequence-specific probes work? | Probe anneals to target DNA (ssDNA) |
| 2 Types of sequence-specific probes? | 1. Hybridisation-based probes 2. Hydrolysis-based probes |
| How do hybridisation-based probes work? | Fluorescence emitted when probe binds to ssDNA template - fluorescence of free probe is quenched by self-binding |
| How do Hydrolysis-based probes work? | Fluorescence emitted by probe hydrolysis following hybridisation to template & primer extension |
| Example of a hydrolysis-based probe please!! | Taqman probes |
| What is Reverse Transcriptase? What does it do? | An RNA-dependent DNA polymerase that changes RNA into cDNA |
| Uses of RT (2) | 1. Gene expression analysis at mRNA level 2. Quantification of infectious agents |
| qPCR method please? (5) | 1. Extract total nucleic acid from tissue/cells 2. Treat with DNAsel to remove all DNA, leaving only RNA 3. Convert mRNA into cDNA using RT 4. qPCR 5. Analyse resulting data |
| Controls needed for qPCR? (3) | Negative control (omit DNA template) RT-minus Positive control (using genomic/plasmid DNA) |
| Methods for getting quantitative data? (2) | Absolute methods Comparative methods |
| What are absolute methods? | Based on actual measurements - standard curve based |
| What are comparative methods? | Standard curve-based - standards may be plasmids with known concentrations - comparative Ct method |
Created by:
rubyroo
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