Enzyme and Restriction Mapping

Question 1

Name three examples of recombinant proteins

Answer 1

Insulin
Interferon
G-CSF

Question 2

Give examples of two transgenic organisms

Answer 2

Disease models
e.g. diabetic mice

Improved agricultural yields
e.g. GM crops

Question 3

What is the use of interferon?

Answer 3

Used as an antiviral agent given to hepatitis C patients

produced by cells infected with viruses

Question 4

What is the use of G-CSF?

Answer 4

growth factor given to patients undergoing radiotherapy to stimulate growth of bone marrow and blood cells

Question 5

What are nucleases?

Answer 5

Enzymes that degrade nucleic acids by hydrolysing phosphodiester bonds

Question 6

Outline the roles of different nucleases

Answer 6

Ribonuclease (RNase): degrade RNA
Deoxyribonuclease (DNase): degrade DNA

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

Question 7

What are restriction endonucleases?

Answer 7

Many different enzymes originating from different bacteria

Question 8

What is the role of restriction endonucleases?

Answer 8

limit transfer of nucleic acids from infecting phages (viruses that affect bacteria) into bacteria by cleaving the phages into smaller pieces

Question 9

What are the 2 main functions of a restriction endonuclease?

Answer 9

1. recognise a specific sequence
2. cut that sequence by catalysing the hydrolysis of
   phosphodiester bonds

Question 10

What is the significance of restriction endonucleases?

Answer 10

Different restriction enzymes recognise different specific DNA sequences

Question 11

What are recognition sequences?

Answer 11

Recognition sites (aka recognition sequences) are 4-8 base pairs in length, depending on the enzyme, and palindromic

Question 12

What is meant by the term palindromic?

Answer 12

A region of DNA in which the sequence of nucleotides is identical with an inverted sequence in the complementary strand

GAATTC is a palindrome of CTTAAG

Question 13

How often do recognition sequences occur in DNA?

Answer 13

In theory:
A 4 base recognition sequence occurs every 4x4x4x4 = 256 bases

A 6 base recognition sequence occurs every 4x4x4x4x4x4 = 4096 bases

Question 14

What are the products of nuclease actions?

Answer 14

Some nucleases produce an overhang either on the 5’ strand or 3’
Some nucleases produce a blunt end

Question 15

What is a restriction map?

Answer 15

A map of an unknown segment of DNA by breaking it into pieces using restriction nucleases and then identifying the locations of the breakpoints

Question 16

Why do we use restriction mapping?

Answer 16

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

Question 17

How would you identify 13kb plasmids (pDTL3) of an unknown molecule without a map?

Answer 17

1. Digest the plasmids using 3 separate tubes of restriction
   nucleases (Eco RI, Bam HI, Eco RI+Bam HI)
2. Run these through gel electrophoresis
   (separating DNA fragments via charge)
3. Place the fragments in the wells at the top of the gel
4. Apply current to enable DNA to migrate to the positive
   pole

Question 18

In Gel Electrophoresis how do we differentiate between fragments?

Answer 18

Migration is faster if the molecule is smaller as it doesn’t get entangled in the mesh of the gel

Question 19

What is the size standard?

Answer 19

A collection of DNA fragments of known size

allows us to compare unknown samples to identify their length

Question 20

What is double digestion?

Answer 20

Digesting a DNA substrate with two restriction endonucleases simultaneously (double digestion) is a common time-saving procedure