L9 Real-Time PCR (Q-PCR)

Question 1

Q-PCR

Answer 1

used for gross estimates of mRNA abundance
very accurate and precise
No gels required, immediate results
Uses fluorescent reporter to detect PCR products

Question 2

Chemistry of Q-PCR

Answer 2

PCR product size : 75.200bp
components are the same as normal PCR
Fluorescent reporter is used

Question 3

DNA binding dye

Answer 3

Sybr Green
Binds non specifically to double stranded DNA and its fluorescence increases up to 10000 fold
Unbound sybr green has little fluorescence.

Question 4

Dye labelling primers: Taqman assay

Answer 4

fluorescent labelled oligonucleotide added in reaction
Taqman binds to complimentary nucleotides
When DNA polymerase extends to where the Taqman is bound, Taqman is degraded, it is broken down and fluorescent is seen

Question 5

Difference between PCR and Q-PCR machine

Answer 5

Contains different filters

Needs to use a transparent seal to detect fluorescence

Question 6

The principle of the quantification

Answer 6

Stage 1: Early stage, little flourescence
Stage 2: cT acceleration of fluorescence
cT is proportional to the inverse of concentration

Question 7

Data analysis: standard curve

Answer 7

cT versus log concentration gives a straight line

Hence, multiple values of cT is needed

Question 8

Reference gene

Answer 8

As with RT-PCR, an internal control is used
Usually a reference gene that is constantly expressed independently.
E.g Eukaryotic: Beta-actin, GAPDH
Prokaryotic: 16S rRNA, sigma 70

Question 9

Quality control of Q-PCR

Answer 9

Melting temp. analysis: temp at which 50% of the double strand DNA is denatured into single strands, release fluorescent dye. Specific to each DNA sequence. Every DNA molecule will DNA at unique temperatures dependant of the sequence.