Enzyme and restriction mapping

Question 1

What type of proteins is genetic engineering use to produce?

Answer 1

• Recombinant proteins
  - Insulun
  - Interferon
  - G-CSF
• Transgenic organisms
  - Disease models
  - Improved agricultural yields

Question 2

What do nucleases degrade and how?

Answer 2

Degrade nucleic acids by hydrolysing phosphodiester bonds

Question 3

What does ribonuclease degrade?

Answer 3

Degrades RNA

Question 4

What does DNase degrade?

Answer 4

Degrades DNA

Question 5

What does exonuclease degrade?

Answer 5

Degrade from the end of the molecule

Question 6

What does endonucleases degrade?

Answer 6

Degrade within nucleotide chain

Question 7

What are the 2 things that restriction endonucleases do?

Answer 7

1. Recognise a specific sequence

2. Cut that sequence

Question 8

Physiology behind restriction

Answer 8

Limit transfer of nucleic acids from infecting phages into bacteria

Question 9

Where are the many different enzymes of restriction endonucleases from?

Answer 9

Many different enzymes from different bacteria

Question 10

What can some nucleases produce?

Answer 10

Some nucleases produce an overhang and some produce a blunt end

Question 11

What are restriction maps?

Answer 11

Map of restriction sites within a molecule

Question 12

What way is useful for describing plasmids?

Answer 12

Restriction maps are a useful way of describing plasmids

Question 13

Restriction enzymes in molecular diagnostics

Answer 13

Sickle cell anaemia:

• Single point mutation
  - A–>T resulting in Glu–>Val

Question 14

What can single nucleotide changes do?

Answer 14

Single nucleotide changes can create/destroy restriction enzyme sites

Question 15

What does DNA ligase do and how?

Answer 15

-Puts 2 fragments together

Does this by repairing nicks in phosphodiesterase backbone

Question 16

What does DNA polymerase do?

Answer 16

DNA synthesis in 5’ to 3’ direction

Question 17

What are the uses of DNA polymerase?

Answer 17

• PCR amplification
• Generation of probes
• Blunt ending of DNA overhangs

Question 18

What do phosphatase’s do?

Answer 18

Hydrolyses a phosphate group off its substrate

Question 19

What are phosphatase’s used to prevent?

Answer 19

Used to prevent cut plasmids from resealing

Question 20

What do polynucleotide kinases do?

Answer 20

Adds phosphate to 5’ hydroxyl group of DNA or RNA

Question 21

What are polynucleotide kinases used for?

Answer 21

• To phosphorylate chemically synthesised DNA so that is can be ligated to another fragment
• To sensitively label DNA so that it can be traced using:
  - Radioactively labelled ATP
  - Fluorescently labelled ATP

Question 22

What are probes?

Answer 22

Fragments of ssDNA

Question 23

What are probes complementary to?

Answer 23

Probes are complementary to the gene of interest

Question 24

What type of enzyme is a reverse transcriptase?

Answer 24

A RNA dependent DNA polymerase

Question 25

How are RNA transcriptase collected?

Answer 25

Isolated from RNA-containing retroviruses

Question 26

What does RNA transcriptase facilitate in the synthesis of?

Answer 26

Synthesises a DNA molecule complementary to a mRNA template using dNTPs

Question 27

What does reverse transcriptase require?

Answer 27

Requires primers

Question 28

Priming for Reverse transcription (Random primers)

Answer 28

Cover all of the length of all of the RNA molecule

Question 29

Priming for reverse transcription (Oligo(dT))

Answer 29

Useful for cloning cDNAs and CDNA libraries, but some might not be full length