Quantification

Question 1

What is Quantification?

Answer 1

Measuring the amount of DNA present in extracted material.

Adding the optimum amount of DNA to your later PCR reaction.

The quantity of DNA from extraction varies significantly on the sample type.

The most used form of quantification is Real-time PCR (qPCR)

Question 2

What are the different types of quantification?

Answer 2

Real-time PCR, Agarose gels, Ultraviolet Spectrophotometry, Fluorescence spectrophotometry and Hybridisation.

Question 3

Agarose Gels

Answer 3

Quick and easy.
Not very accurate.
Better for qualitative analysis as DNA is visualised via the gel under a UV light. (260nm)

Question 4

Ultraviolet Spectrophotometry

Answer 4

DNA absorbs light maximally at 260nm
This method estimates the amount of DNA in an extract by measuring the range of wavelengths between 220 and 300nm.
Then measures absorbance.

Question 5

Fluorescence Spectrophotometry

Answer 5

An alternative to UV Spec.
Specific to double stranded DNA.
Higher sensitivity than UV spec.

Question 6

Hybridization

Answer 6

DNA is applied to positively charged nylon membrane, which is then exposed to a human specific probe.
The emission of the probe is compared to known concentrations.

Question 7

Real-Time PCR (qPCR)

Answer 7

Method used for the detection and quantification of amplified PCR product

PCR product is detected by changes in fluorescence associated with a hydrolysis probe.

Signal increases in direct proportion to the amount of PCR product produced and is monitored at each cycle in real time.