Enzyme and Restriction Mapping

Question 1

List some examples of recombinant proteins.

Answer 1

• insulin
• interferon
• G-CSF

Question 2

The capacity we’ve got to manipulate DNA has resulted in the production of transgenic organisms.
List how those have been used.

Answer 2

• disease models

- improved agricultural yields

Question 3

What are nucleases? What are the two types of nucleases?

Answer 3

They are enzymes that degrade nucleic acids by hydrolysing phosphodiester bonds.

There are two types:
RIBONUCLEASE (RNAse): degrade RNA

DEOXYRIBONUCLEASE (DNAse): degrade DNA

• Exonuclease: degrade from end of molecule
• Endonuclease: cleave within the nucleotide chain

Question 4

Describe restriction endonucleases.

Answer 4

Their main function is restriction: they limit the transfer of nucleic acids from infecting phages into bacteria.
There are many different enzymes from different bacteria.

They do two things:

• recognise a specific sequence
• cut that sequence

Question 5

What are some features of recognition sites?

Answer 5

Recognition sites are 4-8 base pairs in length, depending on the enzyme, and are palindromic.

While some nucleases produce an overhang, some nucleases produce a blunt end.

Question 6

Describe restriction maps.

Answer 6

It is a map of restriction sites within a molecule.

It is a crude way of mapping an unknown molecule, and a useful way of describing plasmids.

Question 7

Can DNA restriction be reversed?

Answer 7

DNA molecules from different sources can be joined together.
If their overhangs are compatible, we would put them together with DNA ligase (whose function is to create phosphodiester bonds between the fragments).

Question 8

What are some uses of DNA Polymerase?

Answer 8

• PCR amplification
• generation of probes
• blunt-ending of DNA overhangs

Question 9

Another enzyme used is phosphatase.

What is a phosphatase?

Answer 9

It is an enzyme that hydrolyses a phosphate group off its substrate.

Question 10

Why would we use a phosphatase?

Answer 10

To prevent cut plasmids from resealing.

Question 11

Another enzyme used is polynucleotide kinase.

What is the function of this enzyme?

Answer 11

It adds a phosphate group to the fragment of DNA. Its function is the opposite of phosphatase.

Question 12

Why would we need to use a polynucleotide kinase?

Answer 12

• to phosphorylate chemically synthesised DNA so that it can be ligated to another fragment
• to sensitively label DNA so that it can be traced using:
  
  • radioactively labelled ATP
  • fluorescently labelled ATP

Question 13

Define a probe.

Answer 13

It is a fragment of ssDNA (or RNA).
It is normally around 20-1000 bases in length.

It is complementary to the gene of interest.

Question 14

Yet another enzyme we use is reverse transcriptase.

What is its function?

Answer 14

It is an RNA-dependant DNA polymerase. It is isolated from RNA-containing retroviruses.

It synthesises a DNA molecule complementary to an mRNA template using dNTPs.