Pre-HW Week 2 From Dr. LeProust

My Homework

1. What’s the most commonly used method for oligo synthesis currently?

The phosphoramidite method using solid-phase synthesis is the most commonly used approach for oligonucleotide (oligo) synthesis today. This method builds oligos in the 3’ to 5’ direction on solid supports like controlled pore glass (CPG) or polystyrene, allowing automation and high efficiency. It involves four key steps per cycle: detritylation to expose the 5’-OH, coupling with activated phosphoramidite monomers, capping unreacted sites, and oxidation to stabilize phosphite triesters into phosphates. Commercial synthesizers make it scalable from small research batches to large therapeutic production.

2. Why is it difficult to make oligos longer than 200nt via direct synthesis?

Direct chemical synthesis of oligonucleotides longer than 200 nucleotides (nt) is challenging due to cumulative inefficiencies and side reactions in the phosphoramidite method.

3. Why can’t you make a 2000bp gene via direct oligo synthesis?

Direct chemical synthesis cannot produce a reliable 2000 bp gene due to exponential error accumulation and physical limitations in the phosphoramidite process. Direct chemical synthesis can’t make a reliable 2000 bp gene because each added nucleotide has only ~99% success rate. Multiplying that over 2000 steps gives almost zero full-length product. Plus, reagents can’t reach the growing chain well on solid supports, causing more errors and junk.