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Related Experiment Video

Updated: May 7, 2025

A Customizable Protocol for String Assembly gRNA Cloning STAgR

A Customizable Protocol for String Assembly gRNA Cloning STAgR

Published on: December 26, 2018

Inert splint-driven oligonucleotide assembly.

Andrew A Mishin¹, Tobin Groth¹, Richard E Green^1,2

¹Claret Bioscience LLC, 100 Enterprise Way, Suite A102, Scotts Valley, CA 95066, United States.

Synthetic Biology (Oxford, England)

|December 30, 2024

Summary

Splynthesis is a novel one-tube method for oligonucleotide assembly using splint-driven reactions. This technique efficiently connects longer DNA fragments, enabling applications in synthetic biology and gene synthesis.

Keywords:

DNA splints gene assembly gene fragments oligos

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Area of Science:

Molecular Biology
Synthetic Biology
Biotechnology

Background:

Traditional oligonucleotide assembly methods like polymerase chain assembly and ligase chain assembly require short, highly purified DNA fragments.
These methods can be limited by the need for extensive purification and the complexity of handling numerous short oligonucleotides.

Purpose of the Study:

To introduce a new in vitro method for oligonucleotide fragment assembly named Splynthesis.
To provide a more efficient and streamlined approach for assembling larger DNA fragments using a one-tube, splint-driven reaction.

Main Methods:

Splynthesis utilizes standard-desalted 'contig' oligonucleotides (approximately 150 nucleotides) and shorter 'splint' oligonucleotides.
Splint oligonucleotides contain 5' and 3' blocking modifications to prevent unwanted ligation and amplification.
The method employs a one-tube, splint-driven assembly reaction for connecting DNA fragments.

Main Results:

The Splynthesis method was successfully used to assemble a 741-bp gene fragment.
The assembled DNA fragment was verified using standard molecular biology techniques, including long-read Oxford Nanopore sequencing.
The resulting product was confirmed to be cloneable via molecular cloning and Sanger sequencing.

Conclusions:

Splynthesis offers a robust and efficient method for oligonucleotide fragment assembly.
The technique is applicable to various fields, including synthetic biology, directed evolution, and functional protein assays.
Splynthesis may also be adaptable for splint-based ligase chain reaction assays.