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FSCI6370 - DNA > Synthesis > Flashcards

Synthesis Flashcards

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1
Q

How are nucleic acids made?

A
  • nucleosides undergo chemical synthesis to form oligonucleotides
  • oligonucleotides undergo enzymatic synthesis to form polynucleotides
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2
Q

What are oligonucleotides (oligos) /polynucleotides (genes)
- number of bases
- what they are used for

A

oligonucleotides
- < 200 bases
- PCR, profiling, diagnosis, sequencing

polynucleotides
- biotechnology
- protein expression
- vaccines

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3
Q

What are chemical synthesis disconnections?

A
  • usually break bonds between carbon and other atoms
  • phosphate linkage is a good site since then each base can be added step-by-step (target C - O bond where O on phosphate group)
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4
Q

What is solid phase synthesis of oligonucleotide

A
  • introduce gene to bead (glass or polystyrene) one at time onto surface of bead until we have the primary structure
  • then cleave from bead at end and we have nucleic sequence that we can do what we want with
  • can scale up with multiple beads
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5
Q

Why is a bead used in solid phase synthesis of oligonucleotides?

A
  • to perform each step in solution would require a purification step at each stage
  • easier to put on bead so do not have to purify at each stage
  • by attaching one end of strand to solid support the excess reagents and side products can simply be washed away
  • beads have little cavities on surface of bead to maximise surface area to be able to have as many interactions as possible
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6
Q

Is solid phase synthesis of oligonucleotides done manually or automatically?

A
  • it is done automated in benchtop machine in lab
  • automation allows us to scale this up
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7
Q

What is synthetic cycle of solid phase synthesis oligonucleotides?

A

1 - couple then wash
2 - cap then wash
3 - oxidise then wash
4 - deprotect then wash

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8
Q

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

A
  • attachment of nucleosides
  • use DMT as protecting group to prevent further reactions and terminate everything
  • the efficiency of this reaction defines how well we are at making target gene
  • if 1 in 200 beads fail then yield of 50 bases is only 78.2 %
  • if 20 in 200 beads fail then yield of 50 bases is only 0.6 %
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9
Q

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

A
  • must catch failed strand as do not want to produce something we don’t want
  • might be dangerous protein so important to do capping to delete mutations and catch them at a time it is still relevant
  • capping = killing unreacted strands
  • 1 coupling failure every 200 strands
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10
Q

What happens if the unreacted strands aren’t capped during the second stage of solid phase synthesis of oligonucleotides?

A
  • leads to deletion mutations
  • primer may not bind
  • error will be carried forwards
  • other biochemistry altered
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11
Q

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

A
  • iodine is oxidising agent
  • THF solvent used
  • water and pyridine also required
  • P=O bond obtained and pyridium iodide formed as side product
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12
Q

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

A
  • removal of DMT using dichloroacetic acid in dichloromethane
  • as do not want this on final synthetic of base pairs
  • trityl cation is stabilised by resonance – making it easy to remove
  • deep yellow/orange colour used to measure coupling efficiency
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13
Q

What are two ways to purify oligonucleotides?

What does this involve?

A
  • PAGE purification
  • HPLC purification
  • failed strands have to be removed in either one of these two ways
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14
Q

What is PAGE purification?

A
  • we know number of base pairs we want
  • run a gel and find length that we want for primary structure
  • find this section in gel, cut out or take it out (freeze and squeeze)
  • older approach
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15
Q

What is HPLC purification?

A
  • allows us to select whatever we want depending on column
  • normal phase - polar molecules will stick to column
  • reverse phase - non-polar molecules will stick to column
  • leave DMT (trityl) group on - very hydrophobic so any group with it on is easy target
  • easy to separate full length strands from failed
  • once separated needs to be held at a stable pH in solution and then freeze dried for storage as solid
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16
Q

What is required to get oligonucleotide to gene?

A
  • maximum length can chemically synthesise from oligonucleotide is 200 bases and typical length of chemically synthesised oligonucleotide is 50 bases so need to do a lot to get full length gene
  • need to get full length gene…
  • …at commercial scale
  • …into RNA (we create DNA but we need RNA)
  • …stable enough to be effective in the body
17
Q

What are two forms of gene synthesis?

A
  • ligation
  • polymerase cycling assembly
18
Q

What is ligation in gene synthesis?

A
  • use enzyme ligase to bridge between pairs to join fragments together to make a single gene (ligase bridges between phosphate groups)
  • need to do PCR amplification to increase size
  • good technique as not many errors (controlled and precise) as all bases of final gene is chemically synthesised
  • suitable for <2k base genes as relies on chemical synthesis
  • only certain limited number of uses of how it can be applied at this level
19
Q

What is polymerase cycling assembly in gene synthesis?

A
  • get fragments to offset together with gaps of 20-30 bases that aren’t artificially synthesised
  • enzyme polymerase fills in the gaps between fragments
  • as we have slightly less control we get more errors e.g. base pairs that are wrong
  • have to cut off point of an error with enzymes (cut of enzymatic fragmentation at error side)
  • melt, anneal, and polymerase to produce target gene
  • less chemical synthesis so longer genes are possible >2k base genes but more problems and potentials for error
20
Q

How is polymerase used in body?

A
  • involved in making and fixing DNA
  • it identifies end of DNA that needs fixing and adds nucleotides to 3’ end of DNA
21
Q

After gene synthesis how do we purify and scale up?

A
  • insert target gene into plasmid
  • transform into bacteria
  • plate and grow
  • sequence colonies (ones that have managed to grow without inserting lots of defects)
  • not too good at being reproducible so must check got what need
  • extract relevant DNA from positive colonies to give us target DNA of what we are interested in
22
Q

How to produce RNA vaccines?

A
  • linearise and trim plasmid DNA to get linear DNA
  • linear DNA undergoes transcription to form RNA
  • put RNA in shell of lipids to protect it from body
  • RNA is what goes into body - to reprogram cells to allow us to fight stuff e.g. covid

FSCI6370 - DNA (16 decks)

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