Synthesis and Purification in a Single Column on a High-Throughput Automated Oligonucleotide Production System

Oligonucleotides are synthesized and purified in a single column filled with a mixture of nucleoside-loaded and underivatized, high-cross link, non-swelling polystryene beads. A new oligonucleotide production system has been developed to completely automate-the entire process of sequence entry, phosphoramidite synthesis, cleavage, deprotection, purification, quantitation, and sample collection. Up to 48 primer-length, high-purity oligonucleotides can be produced in a 24 hour period of unattended automation on the ABI 3948 DNA Synthesizer. Synthesis, cleavage, and purification occur within the unique OneStepTM column. Analyses by MicroGel capillary electrophoresis, anion-exchange HPLC, PAGE, and enzymatic digestion/HPLC routinely exhibit > 90% product purity. Stringent PCR, automated fluorescent sequencing, and genetic analysis experiments show the positive effects of the total automation of purified oligonucleotide production.