DNA Quantification

Question 1

Common Methods of DNA Extraction

Answer 1

1. Agarose Gel Electrophoresis
2. Spectrophotometry
3. Slot blotting
4. Picogreen Microtitre Assay
5. Real time PCR

Question 2

What is Agarose?

Answer 2

Uncharged polymer extracted from seaweed.

Question 3

Agarose Gel Electrophoresis

Answer 3

• DNA loaded into wells and migrates from negative to positive poles.
• Larger fragments move slower than small ones.
• DNA can be visualised by staining with fluorescent dyes which intercalate into DNA. (GelREd or Ethidium Bromide (toxic))
• DNA bound to DNA fluoresces under UV.

Question 4

Loading Dye

Answer 4

• Loading dye (bromophenol blue) added to colourless DNA solution making gel loading easier
• Also indicates the distance travelled by DNA.
• Helps DNA sink into well.

Question 5

Why do some bands fluoresce more on the electrophoresis?

Answer 5

Higher concentration of DNA.

Question 6

Advantages of Agarose Gel Electrophoresis

Answer 6

• Quick and cheap
• Easy to do

Question 7

Disadvantages of Gel Electrophoresis

Answer 7

• Subjective
• Non specific
• Doesn’t work with single stranded DNA.

Question 8

Spectrophotometry

Answer 8

• Measuring absorbance of UV light (260nm) by DNA and using calibration.
• If the machine reads 1.0 then there is 50 micrograms per ml present.
• DNA and RNA have a maximum absorbance of UV light at 260nm.

Question 9

Advantages of Spectrophotometry

Answer 9

• Easy
• Relatively Cheap
• Quick- no prep of material
• Objective- gives number

Question 10

Disadvantage

Answer 10

• Non specific- all double stranded DNA fluoresces
• Does not detect small amounts of DNA
• Contaminants may alter readings.

Question 11

Slot Blotting

Answer 11

• Popular in forensic labs until recently
• Genomic DNA (single stranded) is immobilised on a nylon membrane and subsequently hybridised to a probe (radioactive/chemiluminescent).
• Measurement is made relative to a set of standards (prepared buy serial dilution of a sample of known standards).
• Can detect down to 150 pictograms of DNA

Question 12

Advantages of Slot Blotting

Answer 12

• Sensitive, detects low amount of DNA.
• Single strand specific

Question 13

Disadvanatages of Slot blotting

Answer 13

• Time consuming

Question 14

Picogreen Microtitre Assay

Answer 14

• Highly sensitive fluorescent dye which intercalates into double stranded DNA.
• Unknown samples are compared to known standards.

Question 15

Advantages of picogreen

Answer 15

• High throughput assay in 96-well format possible.
• Sensitive
• Quick and easy

Question 16

Disadvantages of picogreen

Answer 16

• Not specific

Question 17

Real time PCR

Answer 17

• Fluorescently labelled probes or fluorescent dyes are used.
• Fluorescence is measured every cycle.
• Amount of fluorescence is proportional to the amount of PCR product amplified.
• Allows quantification of the amount of template DNA you started out with based on level of fluorescence detected.
• Unknown samples are compared to a set of known standards.
• Most accurate method of DNA Quantification.

Question 18

Disadvantages of real time PCR

Answer 18

Expensive due to machine and kits