Recombinant DNA Technologies

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 (express) a useful protein.

Question 2

How are recombinant DNA technologies useful as fluorescent markers?

Answer 2

If you see the colour then that tells you that the viral vector is being taken up by the cell (as the protein for the fluorescence has been expressed).
If you see nothing it means it is not working.

Question 3

Describe how plasmids are citical elements for recombinant DNA technologies

Answer 3

• Circular pieces of double stranded DNA
• Replicate independently of the host’s chromosomal DNA
• Common in bacteria (but also found in eukaryotes)
• Provide benefit to hosts (eg. antibiotic resistance)

Question 4

What are the key components of recombinant DNA plasmids?

Answer 4

Origin of replication (ORI):
- allows initiation of replication using host DNA polymerase
Antibiotic resistance gene:
- provide survival advantage to cells containing plasmid
Promoter:
- Drives expression of your favourite gene in cells with appropriate transcription factor machinery
Selectable marker:
- to select for cells that have successfully taken up the plasmid (EG. GFP gene (fluorescence))
Restriction sites:
- allows ligation of gene of interest into the cloning vector

Question 5

How do you choose a promotor?

Answer 5

• Promotors are highly specific
• Different transcription factors are found in different cell types and organisms
• The promotor needs to change to allow expression in the difference cell types of the organisms you are trying to express the gene in
• Would need specific vectors
• Cells can replicate a vector, but they will only express the genes if the right transcription factors (in the promotor) are present

Question 6

What is the process for cutting and pasting DNA into plasmids?

Answer 6

• Restriction enzymes are proteins isolated from bacteria that cut DNA
  
  • a bacterial defence mechanism
• Cut dsDNA as specific sequences
  
  • They cut the strands between the same two bases, not just equally in half, this results in over-hanging ends called sticky ends.
    DNA ligase is then added:
• the homologous DNA bases pair up, hydrogen bonds form between the complementary bases
• DNA ligase joins the two stands together, creating a phosphodiester bond between adjacent nucleotides

This is how we make a recombine vector (called that when you have engineered the gene of interest into it)

Question 7

Describe the process of amplifying plasmids (transformation)

Answer 7

• Transformation = transfer of plasmids into bacteria
• Transformed bacteria selected by antibiotic resistance contained on plasmid (so that the only bacteria that survive are the ones that have successfully taken on the plasmid, this means you have a pure sample of bacterial cells that have taken on the vector).
• Expression of plasmid gene in bacteria (if bacterial promotor)
• Amplification of bacteria and purification of DNA for downstream uses (eg. PCR, cloning, transfection into other cells or organisms)

Question 8

Describe how the genetic code is universal and the significance of this

Answer 8

All organisms ‘read’ the same codons as the same amino acids
- AUG = methionine
- UGA = stop codon

Significance: we can transform a human gene into bacteria, and it will still make the same protein

Question 9

What is the only problem with the universal genetic code between organisms?

Answer 9

When cloning eukaryotic genes for expression in prokaryotes, the problem arises of prokaryotes not having introns. This means they don’t have the machinery to remove the introns if they are trying to replicate eukaryotic DNA.
- To resolve this we use cDNA (coding DNA) only! We make cDNA by isolating the fully processed mRNA (which does not contain introns) and then use an enzyme called reverse transcriptase to make that mRNA into DNA again (without introns). This is cDNA.
- Human cells don’t have reverse transcriptase but we have isolated it from other organisms and can react it with the mRNA to do this process of reverse transcription

Question 10

Why use cDNA?

Answer 10

• No introns allows successful translation to a functional protein in prokaryotic cells
• Without the intronic sequence, the overall size of the insert is reduced
  
  • Many vectors have insert size restrictions (so by using cDNA - even if it’s a eukaryotic cell doing the replicating and it doesn’t have the intron issue - it makes the DNA more likely to fit in the vector)