Synthesis

Question 1

How are nucleic acids made?

Answer 1

1. nucleosides go through chemical synthesis to form oligonucleotides
2. oligos undergo enzymatic synthesis to form polynucleotides

Question 2

What is solid phase synthesis of oligonucleotides?

Answer 2

1. introduce gene to a glass bead one at a time until we have the primary structure
2. excess reagents and side products can be washed away
3. then cut off at the bead and we have a nucleic sequence that we can do what we want with

Question 3

What is the bead made from in solid phase synthesis of oligonucleotides?

Answer 3

glass or polystyrene

Question 4

What is the synthetic cycle of solid phase synthesis oligonucleotides?

Answer 4

1. couple then wash
2. cap then wash
3. oxidise then wash
4. deprotect and wash

Question 5

What does DMT do in the coupling stage of solid phase synthesis of oligonucleotides?

Answer 5

stops further interactions

Question 6

What happens in the coupling stage of solid phase synthesis of oligonucleotides?

Answer 6

• attachment of nucleosides
• addition of protecting group DMT

Question 7

What happens in the capping stage of solid phase synthesis of oligonucleotides?

Answer 7

• killing unreacted strands
• 1 coupling failure every 200 strands

Question 8

What happens if the unreacted strands arent capped during the second stage of solid phase synthesis of oligonucleotides?

Answer 8

• leads to deletion mutations
• primer may not bind
• error will be carried forward to protein synthesis
• other biochemistry altered

Question 9

What happens during the oxidation stage of solid phase synthesis of oligonucleotides?

Answer 9

• iodine is oxidising agent
• water + pyridine required to obtain a P=O bond
• THF solvent used
• pyridium iodide side product

Question 10

What happens during the deprotection stage of solid phase synthesis of oligonucleotides?

Answer 10

• removal of DMT

Question 11

What are the two ways to purify the oligonucleotides?

Answer 11

1. PAGE purification
2. HPLC purification

Question 12

What is PAGE purification?

Answer 12

• cut out portion with target strand
• run a gel and find the length that we want and freeze and squeeze then we have the correct lenght of primary structure

Question 13

What is HPLC purification?

Answer 13

• allows selection depending on reverse phase (non-polar stick to column) or normal phase (polar molecules will stick to column)
• leave DMT group on - very hydrophobic so easy to separate full length from failed

Question 14

What is ligation in gene synthesis?

Answer 14

• enzyme ligase joins fragments together
• controlled so there are few errors
• then PCR amplification
• suitable for <2k base genes
• limited by chemical synthesis

Question 15

What are the two forms of gene synthesis?

Answer 15

• ligation
• polymerase cycling assembly

Question 16

What is polymerase cycling assembly in gene synthesis?

Answer 16

• enzyme polymerase fills in the gaps between fragments
• slightly less control so there can be errors
• have to cut off the point of an error with enzymes (enzymatic fragmentation at error site)
• melt, anneal and polymerase to produce target gene
• less chemical synthesis so longer genes are possible - >2000 bases

Question 17

How to form plasmid DNA?

Answer 17

1. insert target gene into plasmid
2. transform into bacteria
3. plate and grow
4. sequence colonies
5. extract DNA from positive colonies

Question 18

How to produce RNA vaccines?

Answer 18

1. plasmid DNA
2. linearise and trim to get linear DNA
3. transcription to form RNA