Synthesis Flashcards
How are nucleic acids made?
- nucleosides go through chemical synthesis to form oligonucleotides
- oligos undergo enzymatic synthesis to form polynucleotides
What is solid phase synthesis of oligonucleotides?
- introduce gene to a glass bead one at a time until we have the primary structure
- excess reagents and side products can be washed away
- then cut off at the bead and we have a nucleic sequence that we can do what we want with
What is the bead made from in solid phase synthesis of oligonucleotides?
glass or polystyrene
What is the synthetic cycle of solid phase synthesis oligonucleotides?
- couple then wash
- cap then wash
- oxidise then wash
- deprotect and wash
What does DMT do in the coupling stage of solid phase synthesis of oligonucleotides?
stops further interactions
What happens in the coupling stage of solid phase synthesis of oligonucleotides?
- attachment of nucleosides
- addition of protecting group DMT
What happens in the capping stage of solid phase synthesis of oligonucleotides?
- killing unreacted strands
- 1 coupling failure every 200 strands
What happens if the unreacted strands arent capped during the second stage of solid phase synthesis of oligonucleotides?
- leads to deletion mutations
- primer may not bind
- error will be carried forward to protein synthesis
- other biochemistry altered
What happens during the oxidation stage of solid phase synthesis of oligonucleotides?
- iodine is oxidising agent
- water + pyridine required to obtain a P=O bond
- THF solvent used
- pyridium iodide side product
What happens during the deprotection stage of solid phase synthesis of oligonucleotides?
- removal of DMT
What are the two ways to purify the oligonucleotides?
- PAGE purification
- HPLC purification
What is PAGE purification?
- 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
What is HPLC purification?
- 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
What is ligation in gene synthesis?
- enzyme ligase joins fragments together
- controlled so there are few errors
- then PCR amplification
- suitable for <2k base genes
- limited by chemical synthesis
What are the two forms of gene synthesis?
- ligation
- polymerase cycling assembly
