Chemical Synthesis of DNA and Gene Assembly 🍠 🌶 Flashcards
Oligonucleotide
a short piece of DNA that is chemically synthesized
How is chemical synthesis done today?
Using an automated DNA synthesizer
Creates DNA by sequentially adding one nucleotide after another in the correct sequence order
What is the direction artificial synthesis is done?
3’ to 5’
What is the first step of artificial synthesis?
Attaching the first nucleotide to a porous material (made of controlled pore glass (CPG)) 🫐
How is the first nucleotide attached?
Not directly, but it is linked to the surface by a spacer molecule that binds to the 3’-OH of the nucleotide
What attaches to the 5’-OH of the nucleotide? What happens because of this?
A chemical blocking group is attached to the 5’-OH so the 3’-OH is the only available reactive group.
Dimethyloxytrityl (DMT) group
The blocking group used in today’s synthesizers
Has an orange color and is easily removed from 5’-OH (so another nucleotide can be linked to the first)
What is important when removing the DMT
Efficiency
(if not removed completely then many of the potential oligonucleotides will fail to elongate)
What is attached to every nucleoside to prevent branching
A di-isopropylamine group attached to the 3’ phosphite group (keeps it stable and allows for long-term storage)
What must happen if the terminal nucleotide fails to react with an incoming nucleotide?
The chain MUST be capped off to prevent the wrong sequence from being generated
What is the finishing step of making artificial synthesis?
All three protective groups must be removed
(DMT groups, cyanoethyl groups, and amino protecting groups)
How does the oligonucleotide become biologically active?
The 5’-OH must be phosphorylated
What efficiency is it critical for DNA synthesizers to have in each round
99% (limits the length of each segment to about 100 bases)
Combining oligonucleotides
Because they are single-stranded, both strands of a gene must be synthesized and annealed together using ligase.
