Real-time PCR

Question 1

meaning of this nomenclature = qPCR

Answer 1

quantitative PCR or Real-time PCR

Question 2

meaning of this nomenclature = rtPCR

Answer 2

reverse transcriptase PCR

Question 3

describe how does qPCR work

Answer 3

• Rxn contains dye - fluorecence when amplified DNA is present
• Thermacycler can read fluorescence generated @ each PCR cycle
  = fluroescence generated = (exact) amount of DNA

Question 4

How does DNA binding dyes (like SYBR green) work, include adv & disadv.?

Answer 4

• non-specific binding to dsDNA
• fluorescence increases 1000 fold when bound to dsDNA
• ADV: cheaper, faster set up, easier design, can perform melt-curve analysis
• DIS: non-specific, can’t use for multiplex rxns (amplifying more than 1 DNA)

Question 5

How does hydrolysis probes (aka TaqMan probes) work, include adv & disadv.?

Answer 5

• 2 molecules = fluorescent/reporter + quencher
• when 2 molecules are in close proximity = no light is emitted bc energy is transferred to quencher
• when separated = light is emitted from fluorescent molecule
• these 2 molecules are on the ends of a probe - specific to gene of interest
• in DNA amp., DNA pol displaces the 5’ end of probe = displaces reporter = light emitted
• ADV: specificity, can be multiplex
• DIS: cost, complex assay design

Question 6

what is the quantification cycle (Cq)? & it’s relationship w/ DNA

Answer 6

• aka threshold cycle
• the # of DNA produced that is detectable by fluorescence
• Cq inversely proportional to # DNA present
• (DNA is limiting factor not reagents)

Question 7

how to optimise qPCR (w/ set up) (7)

Answer 7

• Good lab technique
• Use hot-start Taq = less likely to miss prime
• [Mg2+] ~3mM
• Amplicon length of 75-200 bp
• Primer design for std PCR = (too short = primer dimer. too long = dec efficiency)
• 40 cycles
• annealing & extension combined at 60ºC = single PCR product
• 100 pg to 1ug of gDNA, 1 pg to 100ng of cDNA

Question 8

describe spacing in a quantification curve

Answer 8

• specing in b/w each quantification curve should be evenly spaced
• determined by equ. 2^n = dilution factor
• n= # cycles b/w curves

Question 9

how do you determine amplification efficiency?

Answer 9

• look @ slope of the curve
• OR equ: E=10^(-1/slope)
• optimal slope = -3.32
• to convert efficiency to % = (E-1) x 100
• acceptable % = 95-100%

Question 10

What does High & low efficiency mean?

Answer 10

• Low bc poor primer design; suboptimal rxn cond

- High bc bad pipetting tech, Amplification non-specific, inhibitor in sample

Question 11

What’s the difference b/w Absolut & relative methods of qPCR quantification

Answer 11

• Absolute: determine exact DNA in starting sample (requires std. curve)
• Relative: determine # DNA present in a sample relative to another sample (test vs control)

Question 12

Describe melt-curve analysis

Answer 12

• @ end of qPCR, samples can be melted/denatured slowly w/ inc temp
• As DNA melts = releases SYBR green
• temp DNA melts depends on nucleotide sequence, length
• used to identify SNP

Question 13

How do you normalise Cq of target gene to reference gene?

Answer 13

1. △Cq = Cq (target) - Cq(reference)
2. △△Cq = △Cq(test) - △Cq(control)
3. Normalised expression ratio = 2^(-△△Cq)