Manipulation of DNA

Question 1

What can be used to concentrate DNA?

Answer 1

Ethanol because DNA is not soluble in ethanol.

Question 2

What are the 5 properties of DNA that can be used to manipulate it.

Answer 2

• High solubility in water and insolubility in ethanol.
• Viscosity
• UV absorption
• Denaturation
• Stability

Question 3

How can viscosity of DNA be reduced?

Answer 3

Through denaturation by increasing the temperature or changing the pH.
By shredding DNA.

Question 4

What can be used to discriminate between DNA and protein and why?

Answer 4

UV absorption.

Aromatic bases of nitrogenous bases strongly absorb UV light, more than proteins.

Question 5

Does UV absorption increase of decrease when the DNA is denatured?

Answer 5

Increased

Question 6

What is the melting temperature of DNA?

Answer 6

The temperature at which 50% of the DNA is denatured. It is characteristic of different DNA molecules.

Question 7

Does DNA denature when a person has a fever? When does it denature?

Answer 7

No

During replication and transcription.

Question 8

Is DNA denaturation reversible?

Answer 8

Yes, through annealing. Cooling cannot occur rapidly.

Question 9

Can RNA-RNA hybrids be formed?

Answer 9

Yes

Question 10

Give an example of preimplantation diagnosis.

Answer 10

Use of fluorescent probes through hybridisation.

Question 11

In gel electrophoresis, what is used for detection of bands?

Answer 11

autoradiography, staining (e.g. ethidium bromide and view under UV)

Question 12

What are the differences between polyacrylamide gels and agarose gels?

Answer 12

agarose gels are extracted from seaweed that separate fragments in the size range ~0.6-25kb with discrimination of ~1% of length.
Polyacrylamide gels are synthetic and are used over the size range 1-250 bp (up to ~1kb) and separate fragments differing by a single nucleotide.

Question 13

Compare the distances travelled by linear, circular and supercoiled DNA.

Answer 13

Smallest- slowest
Relaxed circle- middle
Supercoiled circle- fastest.

Question 14

Why does DNA need to first be linearised before being run through gel electrophoresis?

Answer 14

Gel electrophoresis does not only separate molecules based on size and charge, but also based on shape. Circular DNA can change shapes between relaxed and supercoiled and so the distance travelled by it will be influenced by its shape. Linear DNA is not influenced by its shape to the same degree.

Question 15

Which bonds to restriction enzymes break?

Answer 15

Covalent bonds in the sugar-phosphate backbone.

Question 16

Explain the qualities of sequences targeted by restriction enzymes.

Answer 16

• Specific
• 4-8 bases long
• Palindromic
• Sticky end or blunt ends produced

Question 17

Will a restriction enzyme cut more frequently if their recognition site is shorter or longer?

Answer 17

Shorter, more likely to make up a shorter combination.

Question 18

What are flanking regions in DNA, regarding restriction enzymes?

Answer 18

For less specific restriction enzymes, sometimes, the entire recognition sequence doesn’t matter, only the outer few bases do. Those that matter are the flanking regions.

Question 19

What are the steps in southern blotting?

Answer 19

• DNA fragmentation using restriction enzymes
• Amplification of DNA segments
• Gel electrophoresis
• DNA denaturation
• Blotting
• Hybridisation using RNA or DNA probes
• Signal detection