DNA analysis

Question 1

What are the three main steps required to prepare the DNA or RNA sample?

Answer 1

1. Homogenisation/lysis (breaks down the cell, producing a slurry/mixture of everything)
2. Purification (removes the DNA from the slurry)
3. Precipitation/isolation (precipitates DNA and removes any RNA and proteins)

Question 2

What are the two techniques used for quality control and quantification?

Answer 2

1. Electrophoresis
2. Absorbance (this works because the bases contain benzene rings)

Question 3

Describe how the concentration of purified DNA could be quantified using absorbance techniques.

Answer 3

1. First the DNA must be prepared (homogenisation/lysis, purification and precipitation)
2. The sample is placed in a spectrophotometer curvette and the absorbance of 260 nm wavelengths are measured (this is known as the molar extinction coefficient)
3. This absorbance can be converted to concentration via the Beer-Lambert Law.
4. Absorbance is also measured at 230 nm and 280 nm to calculate the ratio between all three wavelength absorbances. This tells you if the sample is contaminated by substances such as phenol (which absorbs at 230 nm), proteins etc. (want a ratio greater than 1.6). This works because nucleic acids (AMP, GMP, etc.) are all most absorbing at 260 nm.

Question 4

What is the Beer-Lambert Law?

Answer 4

An equation that means you can convert absorbance to concentration:

C = (A x molar extinction coefficient x light pathlength)

This can be simplified by converting the extinction coefficients into standard coefficient multipliers for a 1 cm pathway:

C = (A x standard coefficient multiplier)

Question 5

When should you use DNA electrophoresis instead of absorbance measures?

Answer 5

• RNA contamination can result in over-stimulation of DNA -> this can be detected using ethidium bromide gel electrophoresis (although not necessarily quantified)
• When working with small amounts of DNA, such as purified PCR products or DNA fragments extracted from agarose gels, quantification via agarose gel analysis may be more effective

Question 6

What are the steps of gel electrophoresis?

Answer 6

1. Prepare the gel: 1.5-2% agarose dissolved in a buffer of TAE (a mixture of tris-acetate and EDTA), for example. Add ethidium bromide to a concentration of 0.5 micro-g/ml
2. Cast the gel: Heat the mixture to dissolve the agarose, cool and load into the casting tray
3. Run the gel: Add loading dye to the DNA samples (e.g., glycerol). Insert the gel tray into the apparatus and fill the container with running buffer (TAE). Load samples into the wells along with a marker (closing lid) and run at a voltage of ~1-5V.

Question 6

Why does the concentration of agarose gel matter?

Answer 6

• The concentration affects the separation quality. Higher concentrations allow better analysis of long DNA strands and vice versa

Question 7

What does ethidium bromide do?

Answer 7

It is added to stain the DNA as it is fluorescent under UV light (fluoresces orange wavelengths)

Question 8

Why might ethidium bromide be dangerous?

Answer 8

• It is carcinogenic (as it binds to DNA)

Question 9

In which direction does DNA run during gel electrophoresis? Why?

Answer 9

It runs towards the positively charged pole when exposed to an electric field as the phosphate group of each nucleotide has a negative charge

Question 10

How are the band sizes of DNA calculated?

Answer 10

Using a fragment size:migration distances calibration (using a sample of known band sizes)

Question 11

What influences the DNA migration?

Answer 11

1. The size of the DNA molecules (longer molecules migrate more slowly)
2. The agarose concentration (remember high concentrations are associated with the separation of longer strands)
3. DNA conformation (linear, circular etc.)
4. Voltage applied (higher voltages mean that the rate of travel of larger fragments is reduced, meaning that the difference between larger and smaller fragments is reduced)
5. Presence of ethidium bromide/type of agarose/electrophoresis buffer

Question 12

What can be seen visually which indicate bad quality DNA during gel electrophoresis?

Answer 12

• Long streaks after the bands indicate potential DNA degradation
• Bands seen at the far end of the gel (other side of the wells) may indicate RNA contamination
• Samples with bands present in/next to the wells may indicate that the samples contain impurities

Question 13

What can be used instead of ethidium bromide if it is producing too many bands?

Answer 13

Radiolabelled oligonucleotide probes:
- these are like primers with a length of around 30-50 nucleotides attached to a radioactive phosphorous molecule
- these probes bind to specific sites and can be visualised using a film plate (almost like an x-ray)
- this is possible due to the presence of the radioactive phosphorous