Recombinant DNA Technology

Question 1

What is a restriction endonuclease (RE)

Answer 1

An enzyme that binds to a DNA molecule at a particular recognition sequence and makes a specific double stranded cut at or near that sequence

Question 2

What is the biological synthesis of restriction endonucleases?

Answer 2

-They are part of the restriction-modification system of bacteria that provide protection against invasion by foreign DNA

Question 3

How is non-self DNA recognised in bacteria?

Answer 3

The bacterial DNA is methylated by methyltransferase enzyme and non-self DNA is recognised by its lack of methyl groups and is digested by RE.

Question 4

What do the 3 types of RE do?

Answer 4

-Types 1 and 3 cut randomly around target sequence- limited use, unpredictable, not useful
-Type 2 always cut at same position either within or near recognition site - predictable, useful

Question 5

What is the most commonly used restriction enzyme?

Answer 5

Eco RI - comes from ecoli

Question 6

What are the 2 kinds of ends the restriction enzyme can cut?

Answer 6

Stciky ends
Blunt

Question 7

If the sequence after being cut has its 5’ end overhanging?

Answer 7

Sticky 5’ overhang

Question 8

Can sticky ends stick the molecules back togther?

Answer 8

Yes

Question 9

What are examples of cloning vectors?

Answer 9

Plasmids, bacteriophage, cosmids (artificially created plasmids and bacteriophage) and yeast artificial chromsomes

Question 10

Can cohesive (sticky) ends be joined together?

Answer 10

Yes if the overhang is complimentary - how cloning works
Ligase stitches nucleotides together