21.3 In vitro gene cloning - the polymerase chain reaction

Question 1

abbreviation for the polymerase chain reaction

Answer 1

PCR

Question 2

PCR is a method of

Answer 2

copying fragments of DNA

Question 3

what makes PCR rapid and efficent

Answer 3

it is an automated process

Question 4

what are the 5 things that PCR requires

Answer 4

the DNA fragment (to be copied)
DNA polymerase (joins together nucleotides in a matter of minutes)
primers
nucleotide’s (containing each of 4 bases found in DNA)
thermocycler (a computer controlled machine that varies temperatures precisely over a period of time)

Question 5

1. what special feature does DNA polymerase enzyme called taq polymerase have
2. why is taq useful for PCR

Answer 5

1. thermostable (tolerant to heat)

2. it does not denature during high temperatures used in the process

Question 6

what are primers

Answer 6

short sequences of nucleotide’s

that have a set of bases complimentary to those at one end of each of the two DNA fragments

Question 7

what are the 3 stages of PCR

Answer 7

1. Separation of the DNA strand
2. Addition (annealing) of the primers
3. Synthesis of DNA

Question 8

STAGE 1 Separation of the DNA strand

2 steps

Answer 8

1.DNA fragments, primer and DNA polymerase placed in a vessel in the thermocycler.
2.temperature is increased to 95 C
(causing 2 strands of DNA fragments to seperate due to breaking of Hydrogen bonds.)

Question 9

STAGE 2 Addition (annealing) of the primers

1 step

Answer 9

1.mixture is cooled to 55 C

causing primers to join (anneal) to their complimentary bases at the end of the DNA fragment.

Question 10

Importance of primers

Answer 10

1. primers provide starting sequence for DNA polymerase to begin DNA copying because DNA polymerase can only attach nucleotides to the end of an existing chain
2. primers prevent the two separate DNA strands rejoining

Question 11

STAGE 3 Synthesis of DNA

1 step

Answer 11

1. temperature is increased to 72 C
   (the optimum temperature for DNA polymerase to add complimentary nucleotides along each of the separated DNA strands.)
   it begins at the primer on both strands and adds the nucleotide in sequence until it reaches the end of the chain.

Question 12

2 advantages of in vitro (PCR) cloning

Answer 12

1.it is extremely rapid
(within a matter of hours a hundred billion copies of a gene can be made) particularly useful when only a small amount of DNA is available.

2. it does not require living cells
   no complex culturing techniques requiring time and effort.

Question 13

5 advantags of in vivo cloning

Answer 13

1. useful when we wish to introduce a gene into another organism
2. involves almost no risk of contamination
3. it is very accurate
4. it cuts out specific genes
5. it produces transformed bacteria that can be used to produce large quantities of gene products

Question 14

drawback of in vitro (PCR) cloning

Answer 14

increases the other contaminating DNA. need a very pure sample

Question 15

in vivo cloning advantages explained

1. useful when we wish to introduce a gene into another organism

Answer 15

involves the use of vectors
once we have introduced the gene into a plasmid the plasmid can be used to deliver the gene into another organism
i.e. it can transform other organisms (technique called gene therapy)

Question 16

in vivo cloning advantages explained

2. involves almost no risk of contamination

Answer 16

only genes cut up by the same restriction endonuclease will be able to match and therefore combine with the plasma

Question 17

in vivo cloning advantages explained

3. it is very accurate

Answer 17

although mutations can arise they are very rare

Question 18

in vivo cloning advantages explained

4.it cuts out specific genes

Answer 18

very precise procedure as the culturing of transformed bacteria produces many copies of the specific gene, not copies of the whole DNA sample

Question 19

in vivo cloning advantages explained

5. it produces transformed bacteria that can be used to produce large quantaties of gene products

Answer 19

transformed bacteria can produce proteins for commercial or medical use