De Novo Synthesis of Error-Free Long Oligos

Document Type

Article

Publication Date

10-18-2024

Department

College of Forest Resources and Environmental Science

Abstract

This protocol describes the synthesis of long oligonucleotides (up to 401-mer), their isolation from complex mixtures using the catching-by-polymerization (CBP) method, and the selection of error-free sequence via cloning followed by Sanger sequencing. Oligo synthesis is achieved under standard automated solid-phase synthesis conditions with only minor yet critical adjustments using readily available reagents. The CBP method involves tagging the full-length sequence with a polymerizable tagging phosphoramidite (PTP), co-polymerizing the sequence into a polymer, washing away failure sequences, and cleaving the full-length sequence from the polymer. Cloning and sequencing guided selection of error-free sequence overcome the problems of substitution, deletion, and addition errors that cannot be addressed using any other methods, including CBP. Long oligos are needed in many areas such as protein engineering and synthetic biology. The methods described here are particularly important for projects requiring long oligos containing long repeats or stable higher-order structures, which are difficult or impossible to produce using any other existing technologies. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Long oligo synthesis. Support Protocol 1: Synthesis of polymerizable tagging phosphoramidite (PTP). Support Protocol 2: Synthesis of 5′-O-Bz phosphoramidite. Basic Protocol 2: Catching-by-polymerization (CBP) purification. Basic Protocol 3: Error-free sequence selection via cloning and sequencing.

Publication Title

Current Protocols

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