Gene cloning and transfer (BIOL5)

Question 1

Making DNA fragments:

Reverse transcriptase

Answer 1

Make DNA from RNA

1. mRNA isolated from cells
2. Mixed w/ free nucleotides and reverse transcriptase
3. Reverse transcriptase uses mRNA as template to synthesise new DNA stand

• DNA made called complementary DNA (cDNA)

Question 2

Making DNA fragments:

Restriction endonuclease enzymes

Answer 2

• Restr. endonuc - enzymes that recognise spec palindromic seq and cut DNA at these places
• Diff restr. endonuc cut at diff spec recognition seq - shape’s complementary to enzyme active site
  
  • e.g EcoRI cuts only at GAATTC
• If recognition seq present at either side of DNA, can use restr. endonuc to separate fr rest of RNA
• DNA sample incubated w/ spec restriction enzyme, cuts DNA fragment via hydrolysis
• Sometimes cut leaves sticky ends - tails of impaired bases at ends of fragment
  
  • can be used to bind (anneal) fragment to other DNA w/ sticky ends w/ complementary seq

Question 3

Making DNA fragments:

Polymerase chain reaction (PCR)

Answer 3

1. Reaction mix set up containing
   
   • DNA sample
   • primers - pieces DNA complementary to bases at start of fragment wanted
   • polymerase - enzyme creates new DNA strands
2. Mix heated at 95oC to break H bonds b/w DNA strands
3. Mix cooled at 50-60oC so primers can bind/anneal to strands
4. Mix heated at 70oC so polymerase works
   
   • polymerase lines up free nucleotides alongside each template strand - new complementary strand formed
5. 2 new copies of fragment DNA made, one cycle of PCR complete

• Cycle start again w/ mix heated at 95oC w/ all 4 strands (2 orig. 2 new) used as templates
• Each PCR cycle doubles DNA amount

Question 4

Types of gene cloning

Answer 4

1. In vitro - gene copies made outside of organism using PCR
2. In vivo - gene copies made within living organism by DNA replication

Question 5

In vivo cloning

1) Gene inserted into vector

Answer 5

1. DNA fragment inserted into vector DNA e.g plasmids/bacteriophages
2. Vector cut open using restr. endonuc. used to isolate DNA fragment
   
   • Sticky ends of vector complementary to sticky ends of DNA fragment
3. Vector DNA & fragment mixed w/ ligase - joins sticky ends - ligation.
   
   • New combo of based in DNA (vector+fragment) called recombinant DNA

Question 6

In vivo cloning:

2) Vector transfers gene into host cells

Answer 6

1. Vector w/ recombinant DNA used to transfer gene into host cells
   
   • Plasmid vector - host cells placed in ice-cold CaCl solution; walls more permeable
     
     • plasmids added
     • mix heat-shocked (42oC) to encourage cells to take in plasmids
   • Bacteriophages inject DNA into host to infect
2. Host cells take up vectors containing target gene - transformed

Question 7

In vivo cloning:

3) Identifying transformed host cells

Answer 7

• Marker genes used to identify transformed cells
• Inserted into vectors at same time as cloning
  
  • tranformed cells will contain genes cloned and marker genes
• Host cells grown on agar plates:
  
  • divide/replicate DNA, creating colony of cloned cells
• Marker genes can code for antibiotic resistance:
  
  • grown on agjar containing antibio, so only transformed cells have marker gene will survive/grow
• Can code for fluorescence:
  
  • when agar plate under UV light, only transformed cells will fluoresce
• Identified tranformed cells allowed to grow more, prod many copies of cloned gene

Question 8

Advantagesdisadvantages of in vivo

Answer 8

Advantages:

• Can prod. mRNA and protein as it’s in living cells
  
  • has ribosomes/enzymes needed
• Can prod. modified DNA/mRNA/protein
• Large fragments made
• Cheap

Disadvantages:

• DNA fragment must be isolated from other components
• May not want modified DNA
• Slow - some bacteria grow slow

Question 9

Advantages/disadvantages of in vitro

Answer 9

Advantages:

• Only replicate DNA fragment of interest
  
  • don’t have to isolate DNA fragment from host DNA
• V. fast
• DNA not modified

Disadvantages:

• Replicate small DNA fragment (compared to in vivo)
• May want modified DNA
• Expensive if want lots!

Question 10

Benefits of genetic engineering in argiculture

Answer 10

• Crops altered to give h. yield/more nutrition
  
  • reduces cost/famine/environmental probs of using pesticides

Question 11

Benefits of genetic engineering in industry

Answer 11

Enzymes (catalysts) can be prod. from transformed organisms

Question 12

Benefits of genetic engineering in medicine

Answer 12

• Drugs vaccines made using recombinant DNA (insulin)
  
  • made quickly/cheaply

Question 13

Humanitarians think genetic engineering will benefit humans…

Answer 13

• Reduce risk of famine
• Prod. vaccines for more ppl
• Meds made more cheaply - ppl can afford em!

Question 14

Environmentalists/anti-globalist concerns…

Answer 14

• Potentially damage envrionment e.g dec biodiversity
• As use of tech inc., companies get bigger/powerful
  
  • force smaller companies out of business - harder to compete!