DNA analysis

Question 1

What charge does DNA have?

Answer 1

Negative.

Question 2

Describe the steps in electrophoresis.

Answer 2

1. The DNA preparation is run on a gel
2. An electric current is applied across the gel and the DNA migrates to the positive electrode (anode).
3. The gel is then visualised.

Question 3

What are the 2 types of gel used in electrophoresis?

Answer 3

Agarose and acrylamide.

Question 4

What is agarose?

Answer 4

A polysaccharide from seaweed.

Question 5

Which type of gel gives a high resolution?

Answer 5

Acrylamide.

Question 6

What is acrylamide?

Answer 6

A cross-linked polymer that is highly neurotoxic.

Question 7

With agarose there is a need to estimate the sizes of the fragments as it is such low resolution. Describe how you would do this, give 3 steps.

Answer 7

1. Run a marker collection, e.g. an RE digest where the fragments are known sizes, on a gel.
2. Plot this digest on a graph: the y-axis is log size and the x-axis is distance travelled from the well.
3. Use the standard curve to the size of unknown fragments based on how far they have travelled.

Question 8

What chemical is essential for visualising an electrophoresis gel?

Answer 8

Ethidium bromide.

Question 9

What is ethidium bromide (EB)?

Answer 9

A toxic dye.

Question 10

How does EB work?

Answer 10

It intercalates with the DNA molecules, displaying increased fluorescence when intercalated than when free.

Question 11

What wavelength of UV radiation is absorbed by the DNA and transmitted to EB?

Answer 11

254nm.

Question 12

What wavelength of UV is re-emitted by EB?

Answer 12

590nm.

Question 13

What spectrum is the wavelength of UV re-emitted by EB in?

Answer 13

Red-orange.

Question 14

How is the gel visualised after exposure to UV?

Answer 14

With a transilluminator and polaroid film.

Question 15

There are 3 states for the DNA in a plasmid. What are they?

Answer 15

1. Supercoiled, the natural state.
2. Relaxed, when the DNA has been nicked.
3. Linear.

Question 16

Which DNA state travels the furthest in electrophoresis?

Answer 16

Supercoiled.

Question 17

Which DNA state travels the least in electrophoresis?

Answer 17

Linear.

Question 18

What is Southern Blotting?

Answer 18

The hybridisation of a DNA probe to a DNA sample.

Question 19

Describe the 6 major steps in Southern Blotting.

Answer 19

1. Run a RE digest on a gel.
2. Denature the DNA with NaCl
3. De-purinate the DNA with HCl (optional)
4. ‘Blot’ the gel to transfer the DNA fragments onto a membrane.
5. Add a labelled probe in a buffer solution.
6. Detect the probes (depends on which type of probe is used).

Question 20

Why must the DNA be denatured in SB?

Answer 20

Only ssDNA can transfer to the membrane, the double-strands must be split apart.

Question 21

What is the purpose of depurination in SB?

Answer 21

To remove the purine bases, making the DNA fragments smaller.

Question 22

How does the blotting work?

Answer 22

By capillary action.

Question 23

What is used as a membrane in SB?

Answer 23

Nitrocellulose or nylon.

Question 24

If the probes were radioactive how would they be visualised?

Answer 24

By autoradiography.

Question 25

Give 5 major uses of southern blotting.

Answer 25

1. Checking for gene insertion 2. Finding gene homologues across species 3. Finding clones in DNA libraries 4. Forensics - DNA fingerprinting 5. Pre-natal screening using FISH (not strictly SB but same principle)

Question 26

What is Northern Blotting?

Answer 26

Hybridisation of a DNA probe to an RNA sample.

Question 27

What is the purpose of northern blotting?

Answer 27

Tells you which genes are being expressed in a tissue.

Question 28

What happens to excess probes in SB and NB?

Answer 28

They are washed off by buffer solution.