M+C 9 gene technology

Question 1

what is biotechnology?

Answer 1

the manipulation of organisms to make useful products for our benefit

Question 2

what are the 5 basic steps of biotechnology?

Answer 2

1) DNA isolation of desired material from genomes
2) amplify the gene by PCR
3) get the gene into a useful form - cloning vectors
4) put gene into bacterium
5) selection to identify gene is in bacterium

Question 3

how do you isolate DNA from microbes?

Answer 3

either by heat or alkaline lysis (altering PH disrupting the plasma membrane)

Question 4

2 ways you isolate DNA from high organisms which have nuclear membrane or cell walls?

Answer 4

1) mechanical disruption - centrifuge

2) salt buffer detergent

Question 5

how does extraction using salt buffer and detergent work?

Answer 5

salt precipitates the DNA
buffer keeps all at relavent PH
detergent solubilises membranes

Question 6

what are the 3 steps of PCR and the relevant temperatures?

Answer 6

1) denaturation (94-98 degrees)
2) annealing adding primers
3) extension (72 degrees) - DNA polymerase

Question 7

what are the 5 things that go into a PCR reaction?

Answer 7

1) template DNA
2) primers
3) dNTPs - building blocks
4) buffer
5) taq DNA polymerase

Question 8

what does DNA ligase do and where does it get its energy from?

Answer 8

catalyses the formation of a phosphodiester bond between nucleotides by using energy from ATP

Question 9

where do you get restriction enzymes from?

Answer 9

isolated from bacteria which use them to cut foreign DNA

Question 10

what are the 6 parts of a cloning vector?

Answer 10

1) section where DNA is inserted within lac Z gene
2) lac Z gene to identify if cloning vector has required gene
3) origin of replication - plasmid can be copied
4) genes to identify if the plasmid has been transformed e.g. antibiotic resistance
5) restriction sites so the gene can be easily moved between vectors
6) second origin of replication - allows production of single stranded DNA (rarely used )

Question 11

how do you transform the bacteria?

Answer 11

heat shock them at 42 degrees then left to recover in a luria broth at 37 or can use electric shock

Question 12

describe the process of bacterial selection

Answer 12

selecting for bacteria with clone vector by growing on medium with antibiotic to kill any without vector

Question 13

what are the 3 substance provided on the growth plate for blue/white screening?

Answer 13

1) X gal
2) IPTG
3) ampicillin (antibiotic)

Question 14

what does the lac Z gene produce?

Answer 14

beta galactosidase

Question 15

when beta galactosidase combines with x gal what is the outcome?

Answer 15

produces blue colonies

Question 16

what is the job of IPTG?

Answer 16

gives rise to the mutated lac Z gene which produces inactive beta galactosidase that is only active in the presence of the complementary substrate produced by the lac Z fragment in the cloning vector

Question 17

if a white colony is produced what is the outcome?

Answer 17

the desired gene has been taken up because the lac Z gene is interrupted therefore beta galactosidase is not active and x gal cannot react to form a blue colony