Inert splint-driven oligonucleotide assembly.

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.