RNA-based Techniques

Question 1

What does qPCR do

Answer 1

Quantification

Question 2

Methods for qPCR

Answer 2

TaqMan method
SYBR Green method

Question 3

What are they based on

Answer 3

TaqMan: probe-based
SYBR Green: dye-based

Question 4

Similarities of TaqMan and SYBR Green

Answer 4

They both require ds template

Question 5

TaqMan

Answer 5

-More reliable (depends on enzyme activity)
-5’ end: Reporter (R)
-3’ end: Quencher (Q)
-requires: 2 std target-specific primers, a probe
-R: fluorescent, reports signal as more product is generated
-Q: quenches the signal from R under certain circumstances
-when R&Q bind together: no fluorescent signal from R
-TaqMan probes cannot be extended by Taq polymerase

Question 6

What happens during TaqMan

Answer 6

During annealing phase of PCR, Taq polymerase degrades R, R&Q become apart, signals are obtained, measuring the fluorescein signals helps to monitor # of amplicons

Question 7

SYBR Green

Answer 7

-easy, cheap
-ds DNA binding dye: SYBR Green I: fluorescein dye
-SYBR Green protocol: denaturation, annealing, elongation
-amplification is increased as SYBR Green is increased

Question 8

What happens during SYBR Green

Answer 8

Fluorescein dye binds to ds DNA during elongation, fluorescent signal increases, measuring this signal at the end of every thermal cycle helps to determine the quantity of ds DNA present

Question 9

Pros and cons of TaqMan

Answer 9

+
No detection of non-specific PCR products
Multiplex

-
Expensive

Question 10

Pros and cons of SYBR Green

Answer 10

+
Affordable

-
Detection of non-specific PCR products