Patch oligodeoxynucleotide synthesis (POS): a novel method for synthesis of long DNA sequences and full-length genes

Abstract

Synthesis of long DNA fragments is often associated with mutations and requires multiple DNA manipulation steps. A novel DNA synthesis method, referred to as patch oligodeoxynucleotide synthesis (POS) to assembly long DNA fragments is presented here. This method involves connection of two types of oligodeoxynucleotides: long constructional oligonucleotides (COs) and short patch oligonucleotides (POs). Long COs were connected by a ligase with the aid of POs, which were complementary to both adjacent COs to help remove secondary structures during assembly. The partial double-stranded DNA template that was formed was then amplified by PCR. Accordingly, we synthesized SV40 polyadenylation signal sequences (187 bp), a codon-optimized yellow fluorescent protein gene (678 bp), and Rattus norvegicus catenin β1 (2,352 bp). This presented method can be broadly applied to synthesize DNA fragments of varying lengths with great convenience.

Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently

“…following synthesis they are assembled by either PCR-based 715,716 or ligation-based [717][718][719][720] methods. Variant libraries can be created using this process by encoding mixed base codons on the oligonucleotides and at multiple positions if required.…”

Engineering and characterization of fluorogenic glycine riboswitches

“…The riboswitch library contained 94 constructs, where the corresponding linear cDNAs were assembled through POS ( 20 ). In a POS reaction, four similarly long oligonucleotides were ligated to a single DNA strand by bridging with oligonucleotide connectors.…”

“…With this we aimed to exploit the riboswitch mechanism to control the fluorescence state of Spinach by maintaining the function of the sensor part. A synthetic DNA library encoding for 94 glycine riboswitches with different transmitter was constructed rapidly with patch oligonucleotide synthesis (POS) ( 20 ). All glycine sensor constructs were synthesized and functionally, thermodynamically and kinetically characterized in parallel on a microfluidic screening chip platform.…”

Duplex real-time polymerase chain reaction assay for the detection of human KIPyV and WUPyV in nasopharyngeal aspirate pediatric samples

“…KIPyV and WUPyV primer and probes were described in Table 1. As positive control templates, synthetic double-strand oligonucleotides (BMR Genomics, Padua, Italy) from nucleotide 935 to 1024 (90 bases) for KIPyV and from nucleotide 913 to 1001 (89 bases) for WUPyV were designed [19]. The double-strand oligonucleotides were quantitated by spectrophotometry.…”