Chemical Synthesis of DNA Flashcards
Why chem.synth of DNA is in 3’-5’ direction?
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Due to what the synth methods are differed?
P component introducing method
Basic steps in the synth process?
Preparing protected nucleotide monomers
Linking of Nus in order by a suitable phosphorylation method
Removing protecting grps
What are the characters of protecting grps?
Easy to introduce
Stable under rxn conditions
Readily removable
What are the standard protecting grp for each N base? Give rxns for each? How to remove these?
A - benzoyl at 6-amino grp
G - benzoyl at 4-amino grp
C - iso-butyryl at 2-amino grp
Why T is not protected?
Sss
What are the dna synth building blocks?
Ss
Explain the attachment step of dna synth.
Ss
How dmt is removed?
Cl acetic
Explain the coupling step. what catalyze it?
3’-phosphoramidite
Tetrazole
Show the oxidation step. What is the oxidizer?
Phosphite – oxidation > phosphate
I2
The cycle is repeated until the seq is complete
How the dna is released from support?
By adding dil. NH3
What is done in capping? Why is it done?
Acetylate
5’-OH unreacted > can react and missing base – shortmers
Prevent shortmer accumulation
How dna is isolated & purified?
HPLC
