Molecular cloning

Question 1

What is molecular cloning?

Answer 1

A set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules.

Question 2

What is recombinant DNA?

Answer 2

A sequence of DNA composed of 2 or more different sources - e.g. organisms, synthetic & microorganisms.

Question 3

What is the purpose of molecular cloning?

Answer 3

To isolate a gene or sequence:
- to analyse mutant genes vs wild type
- to make mutations
- express & purify the proteins

Question 4

What is a brief overview of molecular cloning?

Answer 4

• make recombinant DNA
• cut & paste vector to the DNA
• put in host & let replication to occur

Question 5

What is a vector?

Answer 5

• a vehicle to carry your gene

Question 6

What are 3 main parts of a vector?

Answer 6

• restriction enzyme sites
• origin of replication
• selectable marker

Question 7

What happens at the restriction enzymes sites?

Answer 7

• where your gene gets cloned (MCS - multiple cloning site)

Question 8

What happens at the origin of replication?

Answer 8

Independent replication inside the host

Question 9

What happens at the selectable marker?

Answer 9

survival of host cells that carry plasmid

Question 10

What are the cons of using a plasmid?

Answer 10

• small (can’t have large insertions)
• low copy number

Question 11

What 3 things are needed to clone DNA into a vector?

Answer 11

• enzyme
• reagents
• techniques

Question 12

If a phage grown in one bacterial host failed to grow in a different bacterial host it is said to have been what?

Answer 12

Restricted

Question 13

If a phage is able to grow in a new host, it is said to have been what?

Answer 13

Modified

Question 14

What protects DNA from being cut up by a restriction enzyme?

Answer 14

The DNA is methylated

Question 15

How many classes of restriction enzymes are there?

Answer 15

4

Question 16

What do type 1&3 restriction enzymes do?

Answer 16

Cleave DNA at random far away from recognition site

Question 17

What do type 2 restriction enzymes do?

Answer 17

Cut DNA at a defined position

Question 18

What do type 4 restriction enzymes do?

Answer 18

Cleave modified DNA

Question 19

What are the 2 types of restriction endings left?

Answer 19

• overhang
• blunt end

Question 20

What element is required for the catalysis of restriction enzyme once its bound to DNA?

Answer 20

Magnesium

Question 21

Why would you use different restriction enzymes on a vector?

Answer 21

To prevent the sticking of sticky ends

Question 22

What is needed to create a phosphodiester bond in the backbone of the nucleotides that have attached to overhanging ‘sticky’ ends?

Answer 22

DNA ligase

Question 23

How does the DNA ligase mechanism work?

Answer 23

1. AMP transferred to enzyme active site.
2. AMP transferred to the 5’ Phosphate
3. AMP-phosphate bond broken by 3’ OH, forming covalent bond

Question 24

What is the problem with recombinant DNA?

Answer 24

• There may not be convenient restriction sites
• Not enough DNA
• DNA may be mixed in with other DNA molecules

Question 25

What occurs if both of the strands of DNA has a phosphate?

Answer 25

Ligation won’t occur

Question 26

What is the purpose of removing the 5’ phosphate (dephosphorylating the vector)?

Answer 26

Prevent self-ligation of the vector

Question 27

What is a way to prevent an overhang?

Answer 27

Use a restriction enzyme that leaves a blunt end.

Question 28

What are the 2 ways of getting recombinant DNA into a host?

Answer 28

1. Electroporation
2. Chemical transformation

Question 29

What is the process of electroporation?

Answer 29

Brief pulse of high voltage

Question 30

What is the process of chemical transformation?

Answer 30

1. Chemically treated E. coli
2. Subject it to heat-shock
3. Causes cell membrane changes that allow uptake of DNA

Question 31

What is used to kill bacteria that doesn’t have a vector?

Answer 31

Antibiotics

Question 32

Why is screening necessary after antibiotics have been used to kill other bacteria?

Answer 32

As it isn’t always efficient