PCR

Question 1

What are the temperature dependent steps of the PCR reaction?

Answer 1

Denaturation - 95
Annealing- 50-65
Extension- 72

Question 2

What are the effects of amplification?

Answer 2

after 25-30 cycles

exponential phase then platos off when running out of primers/substrate

Question 3

Nucleic acid denaturation?

Answer 3

strands are separated - primers annealed

determine melting temp. for each primer - optimum anneal temp

Question 4

What is the Tm?

Answer 4

temperature at which 50% of a molecule remains double stranded and 50% is single stranded = melting temperature

Question 5

Nucleic acid hybridisation?

Answer 5

annealing temp depends on:

GC/AT composition of primer

Higher GC content = higher Tm for same length primer

Question 6

Things to focus on for primer design?

Answer 6

• specificity
• high efficiency
• no primer-dimers

Question 7

how to calculate Tm?

Answer 7

(2 x A+T) + (4 x G+C)

Question 8

What are advantages of PCR?

Answer 8

DNA/RNA can be amplified

Low amounts of starting material

quantitative detection systems also available

multiple samples & targets can be analysed

Question 9

Quantitative PCR

Answer 9

monitor fluorescence emitted during each cycle - real-time

Gives sensitive & specific way of quantifying initial amount of template

kinetic approach

Question 10

What are the kinetics of PCR?

Answer 10

machine will set a threshold
without template - should not be any PCR product made

PCR product passes threshold

Question 11

What is the Ct value?

Answer 11

cycle at which amount of PCR product passes the threshold

Question 12

How is fluorescence produced in qPCR?

Answer 12

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

Question 13

What are hydrolysis based probes?

Answer 13

fluorescence emitted by hydrolysis of probes

followed by hybridisation to the template DNA & primer extension

Taqman

Question 14

What are hybridisation based probes?

Answer 14

fluorescence enhanced when ribs are base paired with template DNA - changes 3D structure

classified as molecular beacons & FRET probes

Question 15

What is TaqMan?

Answer 15

small PCR product

cleavage mediated by 5’ exonuclease of Taq DNA polymerase

Question 16

what are 2 application of qPCR?

Answer 16

quantification of infectious agents

analysis of gene expression at mRNA level

Question 17

Flowchart of qRT-PCR?

Answer 17

tissue/cells

extract RNA

digest contaminating genomic DNA

copy into cDNA

qPCR

DNA proportional to initial no.s of mRNA transcripts

Question 18

What are characteristics of appropriate standards?

Answer 18

expressed in all cells

same copy no. in all cells

expression not change when conditions of cell growth changed

medium copy number more accurate

Question 19

What are commonly used standards?

Answer 19

GADPH (glycolysis)
Beta-actin mRNA
MHC I mRNA
Cyclophilin mRNA 
mRNAs for some ribosomal proteins
28S or 18S rRNA

Question 20

Why are control important?

Answer 20

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

Question 21

What is the standard curve method (absolute)?

Answer 21

unknown samples - protein analysis reaction

read off conc. from standard curve

Question 22

How to calculate the FOLD CHANGE in target gene/transcript

Answer 22

copy number experimental / copy number control

Question 23

What is the comparative method?

Answer 23

measures a change in levels of DNA

applied to changes in cDNA

data analysis method - determines changes in gene expression

Question 24

What is ddPCR?

Answer 24

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

Question 25

What is isothermal amplification?

Answer 25

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

Question 26

How are viral pathogens detected?

Answer 26

CMV - NASBA assay
HIV-1 - genotyping & viral load
Hepatitis B, C - Roche amplicor
HPV - Digene 
SARS-CoV-2 - LAMP assay kit