Real Time PCR

Question 1

What are intercalating dyes? (example)

Answer 1

dyes that bind to any dsDNA, e.g. ethidium bromide, SYBR green
more than 1 dye molecule can bind to PCR product (longer product = more signal)

Question 2

How can intercalating dye be used for detection of primer-dimers?

Answer 2

Dissociation curve analysis

Question 3

3 main types of probes for qPCR

Answer 3

Hybridizing, hydrolyzing, self-fluorescing

Question 4

What type of probe is a hairpin probe?

Answer 4

hybridizing

Question 5

What type of probe is a scorpion/sunrise probe? How does it work?

Answer 5

self-fluorescing

as primer is replicated by polymerase quencher is separated from fluorophore

Question 6

What type of probe is a Taqman probe? How does it work?

Answer 6

hydrolyzing

probe needs to be digested by Taq polymerase to separate the quencher from the fluorphore

Question 7

Baseline

Answer 7

initial cycles when there is little change in fluorescence

Question 8

Threshold

Answer 8

When the real time product can be detected (signal=noise)

Question 9

Cycle threshold (Ct)

Answer 9

the cycle number when the amount of product crosses the threshold (higher Ct=less starting material)

Question 10

Passive reference

Answer 10

dye that provides an internal fluorescence reference to which the reporter dye can be normalized (ROX)

Question 11

Reporter dye

Answer 11

dye attached to taqman probe. signal indicates amplification

Question 12

Normalized reporter (Rn)

Answer 12

fluorescence from reporter - fluorescence from the passive reference dye

Question 13

ΔRn =

Answer 13

Rn - baseline