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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
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Long Oligos: Direct Chemical Synthesis of Genes with up to 1,728 Nucleotides
The longest oligos that can be chemically synthesized using known methods are typically considered to be 200-mers. Here, we report direct synthesis of an 800-mer green fluorescent protein (GFP) gene and a 1,728-mer Φ29 DNA polymerase gene on an automated synthesizer. Key innovations that enabled the breakthrough include conducting the synthesis on the smooth surface of glass wool or glass bead rather than within the pores of traditional solid supports, and the use of the powerful catching-by-polymerization (CBP) method for the isolation of the full-length oligos from the crude mixture. Conducting the synthesis on smooth surface not only eliminated the steric hindrance that would otherwise prevent long oligo assembly, but also, surprisingly, drastically reduced the errors that commonly occur in traditional oligo synthesis. The long oligos were characterized by cloning followed by Sanger sequencing. We anticipate that the new method for long oligo synthesis will have a significant impact on projects in areas such as synthetic biology, gene editing, protein engineering, and many others.
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- PAR ID:
- 10559355
- Publisher / Repository:
- RSC and ACS
- Date Published:
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Accepted Manuscript1.0
- Posted Content:
- https://doi.org/10.26434/chemrxiv-2024-zb7vk
