PCR - L2 Flashcards
How to ensure good PCR?
Good lab hygiene
Why is PCR important?
Amplifying and manipulating DNA
What do we use PCR in?
Diagnosing genetic diseases, detecting pathogens, forensic analysis of DNA
What are the properties of the DNA used in PCR
Clean and pure genetic material
Inhibitor/contaminant free
High concentration
What occurs in reverse transcriptase PCR
RNA -> cDNA -> PCR
What is the source of RNA?
Gene expression or RNA virus
What enzyme is involved in reverse transcription?
Reverse transcriptase
what is qPCR?
PCR that measures the DNA amplification continously
Compare end point PCR w qPCR
End point - cheaper, semi-quantitative, see results at the end and useful in sequencing, genotyping and cloning.
qPCXR - more expensive, Quantity of PCR is proportional to
amount of template, microarray verification, quality control, and assay validation, SNP genotyping,
copy number variation, viral, quantification, siRNA /RNAi experiments. Measures at exponential phase- more
precise
What are the things required in qPCR?
Good, clean genetic material. Thermos lightcycler. SYBR green/TaqMan. DNA polymerase. dNTP. Buffer. Mg2+.
How does SYBR green work?
Fluorescent, binds to groove of ds DNA = increases fluorescence.
How does TAQMan work?
TAQMan works by using a fluorescently labeled probe that binds to the target DNA during PCR; when the probe is cleaved by the DNA polymerase, fluorescence is emitted, allowing real-time detection of the amplification.
What is the threshold line?
the level of detection or the point at which a reaction reaches a fluorescent intensity above background levels
What is the cycle threshold?
Number of cycles required to amplify RNA to detectable level.
What can the cycle threshold be used for?
Estimating the viral load
