CMB2000/L03 Uses of PCR Flashcards
Why is PCR useful? (2)
Manipulate DNA
Detection of pathogens
Diagnosis of genetic diseases
Detecting genetically modified bacteria
Biotechnology
Give 2 examples of how PCR is used in practice.
Manipulate DNA to create artificial fragments and introduce into host cells & incorporated into genome
Knock out gene - study gene function
Fuse host proteins with GFP (localisation)
Why is RNA required before PCR is conducted?
To carry out reverse transcription first (RT -> DNA)
Describe the steps of reverse transcriptase PCR.
Convert RNA to cDNA using RT, retroviral enzyme that converts RNA to DNA
Amplify DNA by PCR (including qPCR)
What are 2 potential sources of RNA for RT PCR?
Gene expression (mRNA): diseased versus healthy/drug effects/environment changes
RNA virus infection levels (E.g. with COVID-19
Describe the steps of RT PCR. (3)
mRNA replicated with poly DT primer
RT produces cDNA
Amplify DNA by PCR
Compare end point PCR to real time PCR.
EP - cheaper, semi-quantitative at best (band intensity), sequencing, genotyping, cloning, see results at end plateau
RT - more expensive, quantity of PCR proportional to amount of template, quantification of gene expression, microarray verification, SNP genotyping, copy number variation, viral quantification, siRNA/RNAi experiments, measures at exponential phase
Describe the theory of qPCR.
Start with extraction and purification of material
Need a thermos lightcycler
SYBR green or Taq/man (fluorescent)
Master mix
Same as endpoint but each cycle cDNA is double
More cDNA than fluorescence
What is required for qPCR?
Standard curve - known amounts, copy number (serial dilution of known concentration)
Normalisation of data to reference genes in tissue/organism
Expression levels - indirectly measuring amount of starting material mRNA; more means more fluorescence, fewer cycles to get to Ct level
Where does SYBR green bind?
Groove of dsDNA to increase fluorescence
How does TAQ/man dye work?
Uses probes with fluorescence reported and quencher
More PCR product = more fluorescence
What does a switch from end point to real time PCR show?
Expression levels of genes
Where is the area of detection for both RT and end point PCR?
RT - exponential phase
EP - plateau
What are reference genes and why are they needed?
Housekeeping genes
Constant level of expression not affected by external factors
Support PCR results validity
Confirms RNA extraction was efficient
Supports credibility
Give an example of a reference gene.
Beta actin
GADPH
Albumin
18S rRNA
TATA sequence binding protein
