Enzymes and restriction mapping

Question 1

Recombinant proteins

Answer 1

Insulin
Interferon
G-CSF

Question 2

Transgenic organisms

Answer 2

Disease models
Improved agricultural yields
Restriction, ligation & other enzymes

Question 3

Nucleases

Answer 3

• Degrade nucleic acids by hydrolysing phosphodiester bonds
• Ribonuclease (RNase): degrade RNA
• Deoxyribonuclease (DNase): degrade DNA

Question 4

Exonuclease

Answer 4

Degrade from end of molecule

Question 5

Endonucleas

Answer 5

Cleave within nucleotide chain

Question 6

Restriction endonucleases

Answer 6

Limit transfer of nucleic acids from infecting phages into bacteria.

Question 7

What do restriction endonucleases do?

Answer 7

1. recognise a specific sequence
2. cut that sequence
   Restriction enzymes catalyse the hydrolysis of phosphodiester bonds

Question 8

Different restriction enzymes recognise …

Answer 8

Different specific DNA sequences

Question 9

What are recognition sites?

Answer 9

4-8 base pairs in length, depending on the enzyme, and palindromic

Question 10

Some nucleases

Answer 10

Produce an overhang or

Produce a blunt end

Question 11

What are restriction enzymes needed for?

Answer 11

Cloning
Molecular diagnostics
Characterization of plasmids

Question 12

What do restriction molecules allow?

Answer 12

DNA molecules from different souces can be joined

Question 13

Restriction enzymes in molecular diagnostics

Answer 13

Sickle cell anaemia - Single nucleotide changes can create/destroy restriction enzyme sites

Question 14

Ddel site

Answer 14

(5’CTNAG3’) is lost is sickle cell anaemia

Question 15

Restriction maps

Answer 15

• Map of restriction sites within a molecule
• Crude way of mapping an unknown molecule
• Useful way of describing plasmids

Question 16

DNA ligase

Answer 16

Repairs nicks in phosphodiester backbone

Question 17

DNA polymerase

Answer 17

DNA synthesis in 5’ to 3’ direction

Question 18

Why use DNA polymerase?

Answer 18

• PCR amplification
• Generation of probes.
• Blunt-ending of DNA overhangs.
The 2 DNA fragments can now be ligated.

Question 19

Phosphatase

Answer 19

Hydrolyses a phosphate group off its substrate.
• Calf intestinal alkaline phosphatase
• Shrimp alkaline phosphatase

Question 20

Why use a phosphatase?

Answer 20

To prevent cut plasmids from resealing.

Question 21

Polynucleotide kinase

Answer 21

Phosphate from ATP to substrate

Polynucleotide kinase adds phosphate to 5’ hydoxyl group of DNA or RNA

Question 22

Why use a polynucleotide kinase?

Answer 22

• To phosphorylate chemically synthesized DNA
  so that it can be ligated to another fragment.
• To sensitively label DNA so that it can be traced
  using:
  - radioactively labeled ATP
  - fluorescently labeled ATP

Question 23

Probes

Answer 23

• Fragment of ssDNA (or RNA)
• 20 – 1000 bases in length
• Complementary to the gene of interest

Question 24

Reverse transcriptase

Answer 24

• RNA dependent DNA polymerase
• Isolated from RNA-containing retroviruses
• Synthesizes a DNA molecule complementary to a mRNA template using dNTPs

Question 25

Priming for reverse trancription

Answer 25

• Random primers: cDNAs up to 700bp but will cover all of the length of all of the RNA molecules.
• Oligo(dT) primers: useful for cloning cDNAs and cDNA libraries, but some might not be full-length.