W3L6 - Real Time PCR Flashcards
Real Time PCR
Quantitative (aka Quantitative PCR) Performed in a single tube Nomenclature - Real-Time PCR (qPCR) - Reverse transcriptase qPCR - RT-qPCR
qPCR Steps
PCR setup similar to conventional PCR
Reaction contains a dye that fluoresces when amplified DNA is present
Thermalcycler contains optical module that reads fluorescence generated at each PCR cycle
Computer records the results and is used to analyse data
SYBR Green 1
DNA binding dye
Binds non-specifically to dsDNA
Little fluorescence when free in solution
Fluorescence increases 1000-fold when bound to dsDNA
Advantages and Disadvantages of SYBR Green 1
Advantages - cheaper and faster to set up - easier to design assays - can perform melt-curve analysis Disadvantages - non-specific - can't multiplex reactions
Hydrolysis Probes
aka TaqMan probes
Based on principle of Fluorescence Resonance Energy Transfer (FRET)
When a molecule fluoresces, its energy can be transferred to a second molecule without emitting light as long as the two are close
Second molecule is called the quencher
When the molecules are separated, light is emitted from the first molecule
How are Hydrolysis Probes used?
Probe specific to the gene of interest is produced
Reporter at one end, quencher on the other
During DNA amplification, 5’ end of the probe is displaced by DNA polymerase and cleaved by 5’ nuclease activity of the polymerase
Reporter dye is dissociated from the quencher -> fluorescence
Advantages and Disadvantages of Hydrolysis Probes
Advantages - specificity - can be multiplexed Disadvantages - cost - more complex assay design
How to read qPCR Results
At the threshold line the amount of DNA produced, and therefore fluorescence is detectable (this is referred to as the quantification cycle; Cq)
The only limiting factor is the amount of DNA present
Therefore the Cq is inversely proportional to the amount of DNA present
How to Calculate Amplification Efficiency
First get slope of cycle number of each dilution Find gradient of the curve E=10^(-1/gradient) Then put E into this formula: % efficiency = (E-1) x 100
What causes low or high efficiency for qPCR optimisation?
Low - poor primer design - suboptimal reaction conditions High - pipetting technique - amplification of non-specific product - inhibitor in sample
Methods of Calculating the Amount of DNA
Absolute - allows to determine exactly how much DNA is in the starting sample
Relative - determines how much DNA is present in a sample relative to another sample
Melt-Curve Analysis
At end of qPCR samples can be ‘melted’ by slowly increasing the temperature
As DNA ‘melts’, SYBR green is released and therefore fluorescence in the reaction decreases
The temperature at which DNA ‘melts’ depends on its nucleotide sequence, length, etc.
Therefore, the temperature at which the fluorescence decreases in the reaction differs for each oligonucleotide and can be used to identify it
What is qPCR used for?
Pathogen detection
mRNA, miRNA expression
Forensic science
Food authenticity studies
