3.8.4.1 Recombinant DNA technology (cloning DNA fragments)

Question 1

what is the difference between in vivo and in vitro?

Answer 1

• In Vivo - copies of DNA fragments are produced inside living organisms, using a vector to transfer DNA into cell (plasmids with circular DNA or baacteriophages/virus)
• In Vitro - copies of DNA fragments are produced inside of living organisms using polymerase chain reaction (PCR)

Question 2

4 Stages of in vivo replication

Answer 2

1. Modify the DNA fragment
2. Insert the DNA fragment into vector
3. Transform a host cell with the vector
4. Identify transformed host cell

Question 3

In Vivo - stage 1 - modification

Answer 3

• in order for the gene to produce proteins it must have a promoter region and a terminator region

Promoter - where RNA polymerase attaches initiating transcription

Terminator - causes RNA polymerase to detach, ending transcription

Question 4

In Vivo - stage 2 - transferring

Answer 4

• vector DNA cut open using the same restriction endonuclease used to isolate DNA fragment , meaning sticky ends are complementary
• vector DNA mixed together with DNA ligase which anneals (joins) sticky ends of vector with sticky ends of DNA fragment

Question 5

In Vivo - stage 3 - transformation of host cells

Answer 5

• cell membrane must be made more permeable so that the target cell takes up the vector
• Ca2+ ions are added and the cells are heat shocked
• this allows the vector to enter the host cells

Question 6

Issues with transformation (stage 3)

Answer 6

• not all host cells will take up the vector (max 5%)
• some plasmids will re anneal together before the DNA fragment is added
• the DNA fragment anneals to itself rather than the vector

Question 7

In Vivo - stage 4 - identification

Answer 7

• marker genes are inserted into vectors at the same time as the gene to be cloned
• marker genes can code for antibiotic resistance, fluorescent proteins, enzyme which cause colour changes
• host cells grown on agar plates containing antibiotic / substrate or agar plates places under UV lights

Question 8

In Vitro - general information

Answer 8

• DNA fragments increased in number using the polymerase chain reaction (DNA replication)
• can be used to make millions of copies of the DNA fragments in 1hr
• automated cycles
  1. Denaturation (95) 2 . Annealing (65) 3. Elongation (72)

Question 9

Stages of in vitro replication

Answer 9

1. Set up reaction mixture containing the DNA sample, free nucleotides, DNA polymerase and primers
2. DNA mixture heated to 95 c to break H bonds between two strands of DNA
3. Mixture is cooled to 65 c so that primers can bind to start of stand (via H bonds and complementary base pairing
4. The mixture is then heated to 72 c for DNA polymerase to line up free nucleotides alongside complementary template strand and join adjacent nucleotides by phosphodiester bonds

Question 10

Stages of in vitro replication

Answer 10

1. Set up reaction mixture containing the DNA sample, free nucleotides, DNA polymerase and primers
2. DNA mixture heated to 95 c to break H bonds between two strands of DNA
3. Mixture is cooled to 65 c so that primers can bind to start of stand (via H bonds and complementary base pairing
4. The mixture is then heated to 72 c for DNA polymerase to line up free nucleotides alongside complementary template strand and join adjacent nucleotides by phosphodiester bonds

Question 11

What are primers?

Answer 11

small single strands of complementary DNA

Question 12

What is the advantage of using DNA polymerase from bacteria?

Answer 12

can heat to higher temperatures, therefore more kinetic energy so faster reaction