Real Time PCR

Question 1

What are real time PCR chemistries?

Answer 1

these are the methods used to create and detect fluorescence upon DNA amplification.

Question 2

What are the 2 major chemistries?

Answer 2

SYBR green

TaqMan (5’ nuclease) probe

Question 3

What is SYBR green?

Answer 3

It is a fluorogenic dye that exhibits little fluorescence when in solution but emits a strong fluorescent signal upon binding to ds-DNA.

Question 4

When is SYBR used over TaqMan?

Answer 4

• When high specificity is not needed or required
• General screening of targets before moving to probe-based assays
• For high resolution melting curve analysis of SNPs
• More flexibility since it doesn’t require designing a separate probe

Question 5

What are the limitations of SYBR green?

Answer 5

• It binds to any dsDNA so not specific for desired amplified sequence.
• Primer dimers will also generate signals from sybr green
• Requires melting/dissociation curve analysis after PCR

Question 6

What is the use of the MC?

Answer 6

Distinguish between primer dimers and specific product

Question 7

How is the MC initiated?

Answer 7

The machine is programmed to do a MC in which the temperature is raised by a fraction of a degree and the change in fluorescence is measured. At MP, the two strands of DNA separate and fluorescence rapidly decreases.

Question 8

The melting point of a dsDNA depends on what?

Answer 8

Its base composition and length

Question 9

All PCR products from a particular primer pair should all have the same melting point unless

Answer 9

there is contamination, mispriming, primer dimer artifacts or some other problem.

Question 10

What is the quality control measure specific to SYBR green which ensures non-specific amplification is not issue?

Answer 10

Running a melt curve

Question 11

Small peaks with lower Tm usually indicate

Answer 11

Primer dimers

Question 12

this chemistry takes advantage of the natural 5’-3’ exonuclease activity of Taq polymerase when it is building a new strand

Answer 12

Taqman 5’ nuclease probe

Question 13

What is the mechanism of 5’ nuclease probes as chemistries?

Answer 13

As Taq polymerase extends the new strand during PCR, it has the ability to cleave any nucleotide in its way. With 5’ nuclease probe, it is designed to anneal on the template between the forward and reverse primers. Cleavage of part of this probe by Taq polymerase is what produces fluorescence during extension.

Question 14

Green emission of the probe is suppressed by the

Answer 14

quencher

Question 15

When can the probe emit green light?

Answer 15

When quencher is separated from it

Question 16

FRET stands for

Answer 16

Fluorescent resonant energy transfer

Question 17

What does Taqman probe use to inhibit fluorescence till PCR amplification?

Answer 17

FRET

Question 18

Why is Taqman called a hydrolysis probe?

Answer 18

Because it is the cleavage from tan polymerase that cause fluorescence to occur

Question 19

What is the difference between FRET usage in hydrolysis and hybridization probes

Answer 19

Hydrolysis probes use FRET to inhibit fluorescence till amplification, but hybridization probes use FRET to enhance fluorescence.

Question 20

List two other mixed mechanism probes

Answer 20

Molecular beacons

Scorpion probes

Question 21

What are hybridization probes used for?

Answer 21

Light cycler technology

Question 22

What modifications have been done on Taqman to improve it?

Answer 22

Made shorter for it to be more specific.
Minor groove binder (MGB) moiety added
Non fluorescent quencher
Sensitive to single-base matches