Quantification

Question 1

Essential or non-essential?

Answer 1

non essential

Question 2

why do we do quantification

Answer 2

a measurement of the amount and quality of DNA in your extract is desirable

difficult to know how well preserved the DNA is

difficult to know how much cellular material has been recovered

optimum input for PCR

Question 3

what is needed for quantification

Answer 3

reference samples

presence of inhibitors

Question 4

what is zero quant

Answer 4

no DNA found

still do process as might find something

Question 5

agarose gel

Answer 5

used to separate DNA fragments

can be used as basic quantification

SYBRsafe dye intercalates with DNA double helix

shows presence/absence of DNA

shows size of DNA fragments

Question 6

what light and wavelength is DNA visualised in agarose gel

Answer 6

UV light at 260nm

Question 7

what will good and bad quality DNA appear as in agarose gel under UV

Answer 7

good = discrete single band

bad= smear

Question 8

advantages of agarose gel

Answer 8

quick

easy

Question 9

disadvantages of agarose gel

Answer 9

very subjective

not very specific, samples may contain bacterial DNA or RNA

poor sensitivity

Question 10

what kind of quantification is Qubit

Answer 10

fluorescence based

Question 11

Qubit

Answer 11

Fluorescence based quantification

Calibrate system using standards provided

Dye in reagents specifically intercalates with dsDNA and increases fluorescence

At a specific amount of dye, the amount of fluorescence signal from sample is directly proportional to the concentration of DNA in the solution

The Qubit fluorometer can pick up this fluorescence signal and convert it into a DNA concentration measurement using DNA standards of known concentration.

some simple calls involved

Question 12

advantages of Qubit

Answer 12

sensitive (down to 10pg/ul)

dsDNA specific (to double stranded DNA)

Question 13

disadvantages of Qubit

Answer 13

not human specific

Question 14

real-time PCR

Answer 14

analyse PCR products after the amplification is complete (28-30 cycles)

it is possible to monitor the PCR products in real-time, as they are produced

when PCR is in real-time it means quantification

uses the TaqMan assay

as more PCR products are generates, the fluorescence of the sample increases

Question 15

advantages of real-time PCR

Answer 15

highly specific

human specific

able to detect inhibition via internal PCR control

Question 16

TaqMan

Answer 16

another fluorescence-based assay

consists of 2 primers and a probe

as the primers are extended during PCR cycles, they eventually meet the probe

the probe is degraded and quenching of the fluorescence is removed and the fluorescence is detected

Question 17

conclusions

Answer 17

3 methods

• agarose gel
• Qubit
• Real-time PCR

With increasing sensitivity and specificity come increased cost and labour