Restriction Enzymes

Question 1

What are restriction endonucleases?

Answer 1

Restriction endonucleases are proteins that cleave DNA molecules at specific sites, producing discrete fragments of DNA.

Question 2

What is the biological function of restriction endonucleases?

Answer 2

Restriction enzymes are believed to protect cells from foreign DNA by cleaving them.

Question 3

How do organisms protect their own DNA from their restriction endonucleases?

Answer 3

Organisms that make restriction enzymes also makes methylases, which marks its own DNA by adding methyl groups to it, protecting it from cleavage.

Question 4

Why are Type II restriction enzymes more commonly used compared to Type I and Type III.

Answer 4

• Type II restriction enzymes only require Mg2+ and not ATP.
• Type II restriction enzymes have only restriction activity,
• Type II restriction enzymes are predictable and consistent.

Question 5

What is a “sticky” end and what is a “blunt” end?

Answer 5

A sticky end is an end of a DNA that has an overhang, while a blunt end does not have an overhang.

Question 6

Provide 2 examples of RE that produces sticky ends and another 2 that produces blunt ends.

Answer 6

• Sticky ends: BamHI and HindIII

- Blunt ends: AluI and HaeIII

Question 7

What are isoschizomers? Give an example of a pair of isoschizomer.

Answer 7

Isoschizomers are different restriction enzymes that has the same recognition sequence.
Example: SphI and BbuI restriction enzymes.

Question 8

What are the factors that affect RE activity?

Answer 8

• Temperature
• pH
• Methylation
• Ionic Conditions (Mg2+ is required for all RE)

Question 9

What happens if RE is not used at the proper condition?

Answer 9

Star activity: Wrong condition alters the specificity of RE which leads to cleavage at non specific sites.

Question 10

What are restriction maps?

Answer 10

Restriction maps are maps that show the relative location of restriction sites.

Question 11

What are palindromic sequences? Describe the two types of palindromic sequence.

Answer 11

Palindromic sequences are DNA sequences that read the same from both directions.

• Mirror-like palindromic sequences: DNA sequence that reads that same from both direction on a single strand
• Inverted repeat palindrome: DNA sequences that reads the same in both direction and are found in the complementary strand.