Serogroup-level resolution of the "Super-7" Shiga toxin-producing Escherichia coli using nanopore single-molecule DNA sequencing.

DNA sequencing and other DNA-based methods are now broadly used for detection and identification of bacterial foodborne pathogens. For the identification of foodborne bacterial pathogens, taxonomic assignments must be made to the species or even subspecies level. Long-read DNA sequencing provides finer taxonomic resolution than short-read sequencing. Here, we demonstrate the potential of long-read shotgun sequencing obtained from the Oxford Nanopore Technologies (ONT) MinION single-molecule sequencer, in combination with the Basic Local Alignment Search Tool (BLAST) with custom sequence databases, for foodborne pathogen identification. A library of mixed DNA from strains of the "Super-7" Shiga toxin-producing Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, O145, and O157[:H7]) was sequenced using the ONT MinION resulting in 44,245 long-read sequences. The ONT MinION sequences were compared to a custom database composed of the E. coli O-antigen gene clusters. A vast majority of the sequence reads were from outside of the O-antigen cluster and did not align to any sequences in the O-antigen database. However, 58 sequences (0.13% of the total sequence reads) did align to a specific Super-7 O-antigen gene cluster, with each O-antigen cluster aligning to at least four sequence reads. BLAST analysis against a custom whole-genome database revealed that 5096 (11.5%) of the MinION sequence reads aligned to one and only one sequence in the database, of which 99.6% aligned to a sequence from a "Super-7" STEC. These results demonstrate the ability of the method to resolve STEC to the serogroup level and the potential general utility of the MinION for the detection and typing of foodborne pathogens.

Duplex formation of the simplified nucleic acid GNA.

Glycol nucleic acid (GNA) has an acyclic backbone of propylene glycol nucleosides that are connected by phosphodiester bonds. This paper characterizes the duplex-formation properties of this simplified nucleic acid. Although single and multiple GNA nucleotides are highly destabilizing if incorporated into DNA duplexes, the two enantiomeric oligomers (S)-GNA and (R)-GNA form antiparallel homoduplexes that are thermally and thermodynamically significantly more stable than analogous duplexes of DNA and RNA. The salt-dependence and Watson-Crick-pairing fidelity of GNA duplexes are similar to those of DNA duplexes, but, apparently, the 2'-deoxyribonucleotide and the propylene glycol backbones are not compatible with each other. This conclusion is further supported by cross-pairing experiments. Accordingly, both (S)- and (R)-GNA strands do not generally pair with DNA. However, (S)-GNA, but not (R)-GNA, forms stable heteroduplexes with RNA in sequences that are low in G:C content. Altogether, the high stability and fidelity of GNA duplex formation in combination with the economical accessibility of propylene glycol building blocks for oligonucleotide synthesis render GNA an attractive candidate for the design of self-assembling materials. They further suggest that GNA could be considered as a potential candidate for a predecessor of RNA during the evolution of life on Earth.

Photoregulation of DNA polymerase I (Klenow) with caged fluorescent oligodeoxynucleotides.

The DNA polymerase reaction by Klenow fragment (KF) was efficiently regulated with UV light using a 25-mer caged fluorescent oligodeoxynucleotide (CFO) as the template. The CFO was functionalized with a fluorescein reporter (Fl) and photocleavable DABSYL quencher moiety (Dab). With Fl and Dab at adjacent cytidines in the middle at the template, KF was blocked from extending a complementary 12-mer primer. Upon UV photolysis of the DABSYL blocking group under aerobic conditions, fluorescein emission was restored and 50% of the primers were fully extended by KF.

A simple glycol nucleic acid.

A glycol nucleic acid (GNA) with an acyclic propylene glycol phosphodiester backbone forms stable antiparallel duplexes following the Watson-Crick base pairing rules.