Restriction Enzymes + Ligase

Question 1

What are restriction enzymes?

Answer 1

Class of enzymes that cut DNA into various fragments.

Known as ‘scissors’

Question 2

What is another biological term for Restriction enzymes?

Answer 2

Endonucleases

Question 3

Where are endonucleases found?

Endonucleases that we look at in biology

Answer 3

Naturally found in bacterial cells

Question 4

What are viruses that invade bacterial cells called?

Answer 4

Bacteriophages

Question 5

Why are restriction enzymes important to bacteria?

Answer 5

They help form bacteria’s defense mechanism (immune response) against bacteriophages

Question 6

How do endonucleases help fight against bacteriophages?

Answer 6

• When bacteriphages invade bacterial cells, the inject their DNA
• Endonucleases cut up viral DNA so that it cannot be synthesised and create more viruses

Prevent viruses from duplicating

Question 7

What are recognition sequences?

Answer 7

• Each type endonuclease has its own recognition sequence
• The endonuclease cuts DNA only where it finds this recognition sequence
• Sequences are generally 4-6 bases long.

Question 8

How do restriction enzymes work?

Answer 8

• They work by snipping specific locations of DNA
• This location is decided by the presence of a recognition sequence
• They break phosphodiester bonds in phosphodiester backbone.

Question 9

How do restriction enzymes not cut bacteria’s own DNA?

Answer 9

• An enzyme called methylase adds a methyl group to specific nucleotide in recogition sequence.
• Methyl group prevents endonucleases from binding.

Question 10

How many DNA fragments are created if a linear piece of DNA is cut twice?

Answer 10

3 fragments

Question 11

How many DNA fragments are created if a circular/looped DNA piece is cut twice?

Answer 11

2 fragments

Question 12

What are the 2 types of restriction enzymes?

Answer 12

• Those that produce Blunt ends
• Those that produce Sticky ends

Question 13

What are Blunt ends?

Answer 13

Cut made by restriction enzymes that has the properties:
* Symmetrical recognition sequence
* Cut on both strands is at same location (exactly opposite each other)

Question 14

What are Sticky ends?

Answer 14

Cut made by restriction enzymes that has properties:
* Non-symmetrical recognition sequence
* Cut at different locations on DNA (not opposite each other)
* Results in single-stranded overhang called sticky end

Refer to diagrams for better understanding

Question 15

Decide whether the following recognition sequences will create blunt or sticky ends:
* GAATTC
* GGCC
* AGAA
* GCTTATTCG

Answer 15

• GAATTC - Sticky End
• GGCC - Blunt End
• AGAA - Sticky End
• GCTTATTCG - Blunt End

Question 16

Describe how blunt ends connecting works

Answer 16

• Any blunt end can connect with any other blunt end
• Fragments can be flipped horizontally and vertically
• Orientation does not matter

Question 17

Describe how sticky ends connecting works

Answer 17

• Only same stciky ends can connect with each other
• Will connect first through hydrogen bonding between complementary base pairs
• Orientation matters and fragments cannot be flipped anyway

See diagrams to help visualise

Question 18

Describe how using the same endonuclease to cut apart two fragments may be beneficial for sticky end DNA fragments

Answer 18

Using same endonuclease means:
* Same recognition sequence is identified
* Hence cut made will produce same complementary sticky ends
* Through hydrogen bonding, sticky ends can connect
* DNA Ligase can be used to connect the fragments

Question 19

Explain how scientists may prefer to work with restriction enzymes that produce sticky ends rather than blunt ends.

Answer 19

• If scientists want to cut fragments and then join them in a specific order, they would prefer sticky ends
• Joining of sticky ends is more controlled and predictable
• This is because they cannot be rotated or flipped anyway

Question 20

Explain why scientists may not want to work with blunt ends

Answer 20

• Ligation of blunt ends is random
• Any two fragments can join in presence of DNA Ligase
• Thus, less useful for DNA manipulation

Question 21

What is Ligation?

Answer 21

The use of DNA Ligase to join together pieces of DNA to make longer pieces of DNA

Question 22

What is purpose of DNA Ligase?

Answer 22

To join together DNA fragments.
Can be used to help repair and replicate DNA strands

Question 23

How does DNA Ligase join fragments of strands?

Answer 23

• By forming strong covalent phosphodiester bonds between the phosphatediester backbone of DNA fragments.
• Linear or circular fragments can be produced

Question 24

What does RNA Ligase do?

Answer 24

Same as DNA Ligase but for RNA

Question 25

For which organism’s DNA can DNA Ligase be used?

Answer 25

Can be used for any organism and can even join DNA fragments from various organisms

Question 26

What factors help set the correct condition for DNA Ligase to carry out ligation?

Answer 26

• Temperature
• Time
• pH

All factors affecting ezyme function would also apply