Recombinant DNA technology

Question 1

What are recombinant DNA technologies?

Answer 1

Joining bits of DNA together (sometimes from different species). These are then inserted into an organism to produce a useful protein

Question 2

Key element for recombinant DNA technology

Answer 2

Plasmids are circular pieces of double stranded DNA. They replicate independently of the hosts chromosomal DNA. Common in bacteria but also found in eukaryotes. Provide benefits to host

Question 3

Key components of plasmids

Answer 3

Origin of replication : allows initiation of replication using host DNA polymerase
Antibiotic resistance gene : allows selection of cells containing plasmid
Promoter : drives expression of your favourite gene (e.g. insulin or GFP) in cells with appropriate transcription factor machinery

Question 4

What are restriction enzymes?

Answer 4

Naturally found in bacteria - defence system to degrade foreign DNA. Cut dsDNA at specific sequences

Question 5

What is DNA ligase?

Answer 5

Complementary base pairing. DNA ligase catalyses the formation of phosphodiester bond to repair nick in DNA backbone allowing the two pieces of DNA to become one

Question 6

What is transformation?

Answer 6

Transfer of plasmids into bacteria. Transformed bacteria selected by antibiotic resistance contained on plasmid. Expression of plasmid gene in bacteria. Amplification of bacteria and purification of DNA for downstream uses

Question 7

The universal genetic code

Answer 7

All organisms read the same codons as the same amino acids meaning we can transform a human gene into bacteria and it will still make the same protein

Question 8

Cloning eukaryotic genes for expression in prokaryotes

Answer 8

Prokaryotic genes don’t have introns hence they don’t have the machinery to process eukaryotic introns
Use reverse transcriptase to make cDNA which removes introns before splicing