PCR Flashcards
What are the temperature dependent steps of the PCR reaction?
Denaturation - 95
Annealing- 50-65
Extension- 72
What are the effects of amplification?
after 25-30 cycles
exponential phase then platos off when running out of primers/substrate
Nucleic acid denaturation?
strands are separated - primers annealed
determine melting temp. for each primer - optimum anneal temp
What is the Tm?
temperature at which 50% of a molecule remains double stranded and 50% is single stranded = melting temperature
Nucleic acid hybridisation?
annealing temp depends on:
GC/AT composition of primer
Higher GC content = higher Tm for same length primer
Things to focus on for primer design?
- specificity
- high efficiency
- no primer-dimers
how to calculate Tm?
(2 x A+T) + (4 x G+C)
What are advantages of PCR?
DNA/RNA can be amplified
Low amounts of starting material
quantitative detection systems also available
multiple samples & targets can be analysed
Quantitative PCR
monitor fluorescence emitted during each cycle - real-time
Gives sensitive & specific way of quantifying initial amount of template
kinetic approach
What are the kinetics of PCR?
machine will set a threshold
without template - should not be any PCR product made
PCR product passes threshold
What is the Ct value?
cycle at which amount of PCR product passes the threshold
How is fluorescence produced in qPCR?
fluorescent reporters
intercalating dyes - bind to DNA, form complexes that emit increased fluorescence compared to free dye
sequence specific probes - added to reaction in addition to normal primers - tell exactly which PCR product has been produced
What are hydrolysis based probes?
fluorescence emitted by hydrolysis of probes
followed by hybridisation to the template DNA & primer extension
Taqman
What are hybridisation based probes?
fluorescence enhanced when ribs are base paired with template DNA - changes 3D structure
classified as molecular beacons & FRET probes
What is TaqMan?
small PCR product
cleavage mediated by 5’ exonuclease of Taq DNA polymerase
what are 2 application of qPCR?
quantification of infectious agents
analysis of gene expression at mRNA level
Flowchart of qRT-PCR?
tissue/cells
extract RNA
digest contaminating genomic DNA
copy into cDNA
qPCR
DNA proportional to initial no.s of mRNA transcripts
What are characteristics of appropriate standards?
expressed in all cells
same copy no. in all cells
expression not change when conditions of cell growth changed
medium copy number more accurate
What are commonly used standards?
GADPH (glycolysis) Beta-actin mRNA MHC I mRNA Cyclophilin mRNA mRNAs for some ribosomal proteins 28S or 18S rRNA
Why are control important?
negative control - checks for contamination
no reverse transcriptase control - detects if signal from contaminating DNA
positive control - checks reagents & primers work
important if trying to show absence of expression of a gene
What is the standard curve method (absolute)?
unknown samples - protein analysis reaction
read off conc. from standard curve
How to calculate the FOLD CHANGE in target gene/transcript
copy number experimental / copy number control
What is the comparative method?
measures a change in levels of DNA
applied to changes in cDNA
data analysis method - determines changes in gene expression
What is ddPCR?
absolute method
emulsion PCR
after 40 cycle - each droplet analysed for fluorescence in special reader
each droplet contains 1 template and all reagents for qPCR
develop droplets & disperse templates into single droplets - can be amplified
What is isothermal amplification?
everything done at same temp - 42degC
Hybrid primer
RT primer
make second strand
Phage RNA polymerase
detected by fluorescence or enzyme linkage
ALL DONE AT 1 TEMP
How are viral pathogens detected?
CMV - NASBA assay HIV-1 - genotyping & viral load Hepatitis B, C - Roche amplicor HPV - Digene SARS-CoV-2 - LAMP assay kit
