Restriction enzymes

Question 1

20.1 Recombinant DNA Technology Began with Two Key Tools:

Answer 1

Restriction Enzymes and Cloning Vectors

Question 2

Recombinant DNA technology is aka

Answer 2

Genetic Engineering

Question 3

Recombinant DNA technology/ genetic engineering is based on the ______________ of DNA molecules at __________________.
Resulting DNA fragment can be transferred from any organism to a virus or bacterial cell in order to ____________

Answer 3

cutting and pasting
specific base sequences
mass-produce, isolate and characterise it.

Question 4

Recombinant DNA technology/ genetic engineering is used for

Answer 4

gene mapping
diagnosis of diseases
commercial production of human gene products
expression of foreign genes in transgenic animals and plants.

Question 5

Making recombinant DNA
The process comprises a series of steps:

Answer 5

1. DNA is isolated from cells
2. DNA is cut by restriction enzymes (REs)
3. Fragments are ligated into “vectors”
4. Recombinant molecules are transformed into host cells
5. Multiplication occurs in host cells
6. Cloned DNA can be recovered, purified and analysed

Question 6

Function of restriction Enzymes

Answer 6

Cut DNA at Specific Recognition sequences

Question 7

How are Restriction Enzymes produced?

Answer 7

Produced by bacteria as a means to combat bacteriophage infection.
= Degrade the DNA of infecting viral pathogens.
[Endonucleases (antiviral protection against phages)]

Question 8

How do Restriction Enzymes cut DNA sequences?

Answer 8

Cuts both strands of DNA within the recognition sequence by cleavage of the phosphodiester backbone.

Question 9

Some common restriction enzymes, outlining their (palindromic) recognition sequences. Note their diverse DNA cutting patterns, and source microbes. The arrows indicate the site in the DNA where each respective enzyme will cut.

Question 10

Characteristics of restriction enzymes

Answer 10

• Two types: Type I and II
• > 500 commercial REs
• Palindromic recognition sequences
• Generate restriction fragments
• Frequent vs. rare cutters
• Sticky (cohesive) vs. blunt ends

Question 11

Recognition sequences of restriction enzymes occur randomly in the genome as palindromes, what does this mean?

Answer 11

they read the same on both strands of DNA.
Read in a 5’-3’ and a 3’-5’ orientation

Question 12

Recognition sequences of restriction enzymes are named after the

Answer 12

organism from which they are discovered

Question 13

EcoRI was the first restriction enzyme discovered from

Answer 13

Escherischia coli strain R.

Question 14

Depending on their recognition sequences, restriction enzymes can produce either

Answer 14

sticky” or “blunt” ends

Question 15

Sticky ends

Answer 15

Sticky ends create overhangs

Question 16

Blunt ends

Answer 16

create blunt ends

Question 17

Pieces of DNA which have sticky ends generated by the same restriction enzyme are able to

Answer 17

anneal because of complementary base pairing.

Question 18

Pieces of DNA which have sticky ends generated by the same restriction enzyme are able to anneal because of complementary base pairing.
But there are still nicks left after this.

Answer 18

DNA ligase will seal the nick via ligation

Question 19

Explain ligation

Answer 19

DNA ligase will seal the nick in the phosphodiester backbone of DNA by using ATP to catalyze the covalent joining of the two fragments together, forming recombinant DNA molecules.

Question 20

Restriction enzyme EcoRI comes from

Answer 20

Escherichia coli R

Question 21

Restriction enzyme HindIII comes from

Answer 21

Haemophilus influenzae RD

Question 22

Restriction enzyme BamHI comes from

Answer 22

Bacillus amyloliquefaciens H

Question 23

Restriction enzyme TaqI comes from

Answer 23

Thermus aquaticus

Question 24

Question 25

1. What is the palindromic recognition sequence of HindIII?
2. What does the III stand for in HindIII?
3. Does HindIII generate a blunt end or sticky end??

Answer 25

1. AAGCTT
2. It is the third restriction enzyme discovered from H. influenzae Rd
3. Sticky ends

Question 26

Which of the following is a palindromic recognition sequence?
A)5’. . . CAATAG . . . 3’
B) 5’. . . CAATTG . . . 3’
C) 5’. . . CATTTG . . . 3’
D) All of the above
E) None of the above

Answer 26

B

CAATTG
GTTAAC

Question 27

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Question 30