Quantitative PCR Flashcards
What type of approach is real-time PCR?
Kinetic
How does the fluorescent signal produced increase in response to the amount of PCR product produced?
Exponentially
How is the fluorescence generated in real time PCR ?
It is generated by fluorescent reporters: intercalating dyes which bind to DNA and form complexes that emit increased fluorescence in comparison with the free dye, when irradiated with light of the wavelength characteristic for a particular dye, e.g. SYBR Green or appropriate probes.
How does Sybr Green work?
Binds to double stranded DNA but not to single-stranded DNA and is frequently used to monitor the synthesis of DNA during real-time PCR reactions. When it is bound to double stranded DNA it fluoresces very brightly.
What is required in addition to the normal PCR primers in real-time PCR?
Sequence specific oligonucleotide probe(s), complementary to an internal sequence of the amplified fragment.
When is fluorescence emitted using a hydrolysis based probe eg. TaqMan?
By hydrolysis of probes following their hybridization to the template DNA and primer extension.
When is fluorescence greatly enhanced using a hybridisation based probe?
When probes are based paired with the template DNA, which changes their 3D structure. Dependent on their mechanism of action they are classified as: Beacons or FRET probes.
What are the applications of real-time PCR?
Quantification of infectious agents (HIV, HPV), Analysis of gene expression at the mRNA level, Genotyping.
What is real-time PCR?
Form of the polymerase chain reaction that measures the amount of the amplified DNA product by monitoring fluorescence emitted during each cycle (real-time) instead of at a fixed end-point. Provides a sensitive and specific way of quantifying the initial amount of template, provided that appropriate standards are used.
What are some methods of quantification of mRNA?
Northern blotting, ribonuclease protection assay, in situ hybridization and Reverse transcription (RT) - PCR.
Which method of quantitating mRNA is most sensitive and why?
Reverse-transcriptase PCR as it can discriminate between closely related mRNAs and it’s technically simple.
Give the steps in the overlies of reverse transcription- PCR?
1) tissue/cells
2) extract RNA
3) copy into cDNA using reverse transcriptase, oligo dT and random hexamer primers
4) do real-time PCR
5) analyse results
What should the primers be in reverse transcription-PCR?
Specific, have high efficiency, no primer dimers and ideally should not give a DNA signal
How are primers designed for reverse transcription-PCR?
In exons.
What should standards used have?
The same copy number in all cells, expression in all cells, expression that does not change when conditions of cell growth are changes and medium copy number is advantageous.
