21.2 in vivo gene cloning

Question 1

what are recognition sites?

Answer 1

sequence of DNA that is cut by restriction endonucleases

Question 2

why is the same restriction endonuclease used in the plasmid as the DNA?

Answer 2

fragments produced will have sticky ends that are complementary to one another

Question 3

what does DNA ligase do?

Answer 3

joins DNA fragment with plasmid

Question 4

what method do you have to use to prepare the DNA fragment for insertion?

Answer 4

1. add promoter

2. add terminator

Question 5

what is a promoter?

Answer 5

region of DNA that provides a binding site for RNA polymerase to attach

Question 6

what is a terminator?

Answer 6

region of DNA the releases RNA polymerase

Question 7

what is a vector?

Answer 7

carrier of DNA into a host cell

Question 8

what is the most common vector?

Answer 8

plasmid

Question 9

explain the method for inserting the DNA fragment into a vector

Answer 9

1. plasmid and DNA cut using the same restriction endonucleases
2. mix plasmids and DNA together
3. add DNA ligase

Question 10

what is transformation?

Answer 10

process by which plasmids are reintroduced to bacteria cells

Question 11

explain the method for transformation

Answer 11

1. mix plasmids and bacterial cells together in a medium containing Ca2+ ions
2. heat

Question 12

why will only 1% of the bacterial cells take up the plasmids?

Answer 12

• some plasmids will have closed up without incorporating the DNA fragment
• some DNA fragment ends join together to form its own plasmid

Question 13

what are marker genes?

Answer 13

genes used to identify whether a gene has been taken up by bacterial cells

Question 14

what are 3 types of marker genes?

Answer 14

• antibiotic resistant marker
• fluorescent marker
• enzyme marker

Question 15

how do fluorescent markers work?

Answer 15

• if plasmid has been taken up bacteria doesn’t fluoresce

- if plasmid hasn’t been taken up then fluoresces

Question 16

how do enzyme markers work?

Answer 16

• if plasmid has been taken up, bacteria doesn’t produce enzyme
• if plasmid hasn’t been taken up, bacteria produces enzyme

Question 17

how do antibiotic- resistant markers work?

Answer 17

• if plasmid has been taken up, bacteria is resistant to 1 antibiotic
• if plasmid hasn’t been taken up, bacteria is resistant to 0 antibiotics
• if plasmid has been taken up but doesn’t have DNA fragment, bacteria is resistant to 2 antibiotics