Unit 2: Recombinant DNA

Question 1

What do restriction enzymes do ?

Answer 1

They cleave phosphodiester bonds between nucleotides in DNA

Question 2

How many different types of restriction enzymes are there ?

Answer 2

4

Question 3

What type of restriction enzyme is often used in recombinant biology ?

Answer 3

Type 2

Question 4

What is the job of type 2 restriction enzymes ?

Answer 4

They cut beside/ within the middle of the recognition sequence

Question 5

Give 3 features of type 2 restriction enzymes ?

Answer 5

• They recognise 4-8nt double stranded DNA sequences
• Recognition sites are usually palindromic
• Cut within the recognition sequence

Question 6

Give 3 different names of type 2 restriction enzymes

Answer 6

• Bam H1
• Kpn 1
• Sma 1

Question 7

At what position does Bam H1 cut DNA ?

Answer 7

Between bases 1-2 of the palindromic sequence

Question 8

At what position does Kpn 1 cut DNA ?

Answer 8

Between bases 5-6

Question 9

At what position does Sma 1 cut DNA ?

Answer 9

Between bases 3-4

Question 10

What is DNA ligation ?

Answer 10

The re-joining of compatable sticky ends by creating new phosphodiester bonds

Question 11

What is the most commonly used DNA ligase in molecular biology ?

Answer 11

T4 DNA ligase

Question 12

What is the optimum temp for DNA ligase to work ?

Answer 12

37 degrees C

Question 13

What 2 things are required for T4 DNA ligase to work ?

Answer 13

1) 5’ end of DNA to be phosphorylated
2) Requires ATP as part of ligation reaction

Question 14

What is a plasmid ?

Answer 14

Circular pieces of DNA that are independent from the bacterial chromosome

Question 15

What are 3 components of a plasmid ?

Answer 15

• Origin of replication
• Antibiotic resistance gene
• Multiple cloning site

Question 16

Give 2 advantages of cloning into plasmids

Answer 16

1) Enables expression of gene to produce recombinant proteins
2) Generates much more DNA to PCR

Question 17

How are fragments closed into a fragment using a restriction enzyme ?

Answer 17

1) A plasmid and fragment with matching ER sites are chosen
2) They are then incubated (digested) with EcoR1 (RE) and compatible sticky ends are made
3) Ligation creates a new circular DNA plasmid containing a fragment

Question 18

If fragments are cloned with only 1RE what does this mean ?

Answer 18

It means that inserts can be clones into a vector in 2 orientations

Question 19

What is transformation ?

Answer 19

Getting DNA into bacteria