Recombinant techniques

Question 1

define: restrinction endonuclease

Answer 1

enzyme purified from bacteria, which cuts both strands of DNA at specific sties

Question 2

why do bacteria produce restriction enzymes

Answer 2

to degrade incoming viral DNA

Question 3

how are restriction enzymes named

Answer 3

after the bacteria they come from

e. coli –> EcoRI

Question 4

key features of type II restriction endonucleases

Answer 4

usually make cut within or near the recognition site
usually 4-8bases long and palindromic
require Mg2+ but not ATP

Question 5

what’s special about ecoRI recognition site

Answer 5

can’t cut when recognition site contains methylated A

Question 6

describe the process of cloning

Answer 6

RE exposed to target sequence and plasmid

join, sealed with DNA ligase

put into competent bacteria

Question 7

what’s special about the E. coli expression vector

Answer 7

polylinker
selection markers - antibiotic, blue-white selection
transcription: promoter, operator/repressor, termination site
translation: ribosome binding site

Question 8

describe site directed mutagenesis

Answer 8

 DS plasmid denatured
 Primers containing mutation are made, and anneal to the ss plasmids
 DNA p extends mutated primers
 Unmuted parent DNA is degraded with methylation dependent restriction endonucleases (ensures only old, not new, DNA is degraded)
 The 2 ss mutated plasmids anneal; and is inserted into plasmid

Question 9

describe blue-white selection

Answer 9

beta galactosidase

if interrupted no colour will be produced. if uninterrupted, blue.