Inert splint-driven oligonucleotide assembly

Abstract

In this study, we introduce a new in vitro method for oligonucleotide fragment assembly. Unlike polymerase chain assembly and ligase chain assembly that rely on short, highly purified oligonucleotides, our method, named Splynthesis, uses a one-tube, splint-driven assembly reaction. Splynthesis connects standard-desalted "contig" oligos (∼150 nt in length) via shorter "splint" oligos harboring 5' and 3' blocking modifications to prevent off-target ligation and amplification events. We demonstrate the Splynthesis method to assemble a 741-bp gene fragment. We verify the assembled polymerase chain reaction product using standard molecular biology techniques, as well as long-read Oxford Nanopore sequencing, and confirm that the product is cloneable via molecular means, as well as Sanger sequencing. This approach is applicable for synthetic biology, directed evolution, functional protein assays, and potentially even splint-based ligase chain reaction assays.

Keywords: DNA splints; gene assembly; gene fragments; oligos.

Figure 1.

Schematic overview of Splynthesis.

Figure 2.

Assessments of the input contigs and the dsDNA assembly products. (a) Insert distribution plots for each individual contig oligo (Illumina sequenced). (b) Gel image from a D5000 TapeStation trace to visualize PCR cycle titration of the assembly product. (c) Insert distribution plots for each assembly amplicon cycles 15–30 (ONT sequenced). (d) Average ONT base quality score at 741 bp for each assembly amplicon cycles 15–30. (e) Aggregated histogram of ONT CIGAR scores per read at 741 bp for each assembly amplicon cycles 15–30.

Figure 3.

Assessment of the cloned amplicon. (a) Blue/white colony counts for controls and vector ± insert (amplicon). (b) Gel image from a D5000 TapeStation trace to visualize the restriction digest evaluation of minipreps from 20 white colonies. (c) IGV visualization of Sanger sequencing results.