Enzyme and restriction mapping

Question 1

What type of proteins is genetic engineering use to produce?

Answer 1

Recombinant proteins

• Insulin
• 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 are the two types of nucleases?

Answer 3

• RNase = Ribonuclease - degrades RNA
• DNase = Deoxyribonuclease - degrades DNA

Question 4

What is the difference between endo and exonucleases?

Answer 4

o Exonuclease – degrades from the end of the molecule o Endonuclease – cleaves within the nucleotide chain

Question 5

What are the 2 things that restriction endonucleases do?

Answer 5

1. Recognise a specific sequence
2. Cut that sequence

Question 6

Physiology behind restriction

Answer 6

Limit transfer of nucleic acids from infecting phages into bacteria

Question 7

What are features of recognition sites?

What do nucleases produce?

Answer 7

• 4-8 base pairs in length
• depending on the enzyme are palindromic

Some nucleases will produce overhang or blunt ends

Question 8

What are restriction maps?

Answer 8

Map of restriction sites within a molecule

Question 9

Which enzyme is used to convert overhangs into blunt ends

Answer 9

DNA polymerase

Question 10

Restriction site in sickle cell anaemia

Answer 10

In SCA, the restriction site (5’CTGAG3’) for enzyme DdeI enzyme is changed due to a single nucleotide change (A to T), and therefore enzyme can’t recognise sequence and there is no restriction site

Question 11

What does DNA ligase do and how?

Answer 11

• Puts 2 fragments together
• Does this by repairing nicks in phosphodiesterase backbone by forming phosphodiester bonds

Question 12

What does DNA polymerase do?

Answer 12

Synthesises DNA in the 5’ to 3’ direction

Question 13

What are the uses of DNA polymerase?

Answer 13

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

Question 14

What do phosphatase’s do?

Answer 14

Hydrolyses a phosphate group off its substrate

Question 15

What are phosphatase’s used to prevent?

Answer 15

Used to prevent cut plasmids from resealing

• DNA ligase needs phosphate groups

Question 16

What do polynucleotide kinases do?

Answer 16

Adds phosphate to 5’ hydroxyl group of DNA or RNA

Question 17

What are polynucleotide kinases used for?

Answer 17

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 18

What are probes?

Answer 18

• Fragments of ssDNA
• 20-1000 bases in length
• Complementary to gene of interest

Question 19

What type of enzyme is a reverse transcriptase?

Answer 19

A RNA dependent DNA polymerase which makes copies of RNA molecules from DNA

Question 20

Where is reverse transcriptase found?

Answer 20

Retroviruses whose genomes are made of DNA

Question 21

What does RNA transcriptase facilitate in the synthesis of?

Answer 21

Synthesises a DNA molecule complementary to a mRNA template using dNTPs

Question 22

What does reverse transcriptase require?

Answer 22

Requires primers to bind mRNA molecules so that reverse transcriptase can bind

There are different ways to prime reverse transcriptase

• random primer
• oligo(dT) primer
• gene specific primer

Question 23

Priming for Reverse transcription (Random primers)

Answer 23

Cover all of the length of all of the RNA molecule

Question 24

Priming for reverse transcription (Oligo(dT))

Answer 24

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