Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array.

DNA sequencing by synthesis (SBS) offers a robust platform to decipher nucleic acid sequences. Recently, we reported a single-molecule nanopore-based SBS strategy that accurately distinguishes four bases by electronically detecting and differentiating four different polymer tags attached to the 5'-phosphate of the nucleotides during their incorporation into a growing DNA strand catalyzed by DNA polymerase. Further developing this approach, we report here the use of nucleotides tagged at the terminal phosphate with oligonucleotide-based polymers to perform nanopore SBS on an α-hemolysin nanopore array platform. We designed and synthesized several polymer-tagged nucleotides using tags that produce different electrical current blockade levels and verified they are active substrates for DNA polymerase. A highly processive DNA polymerase was conjugated to the nanopore, and the conjugates were complexed with primer/template DNA and inserted into lipid bilayers over individually addressable electrodes of the nanopore chip. When an incoming complementary-tagged nucleotide forms a tight ternary complex with the primer/template and polymerase, the tag enters the pore, and the current blockade level is measured. The levels displayed by the four nucleotides tagged with four different polymers captured in the nanopore in such ternary complexes were clearly distinguishable and sequence-specific, enabling continuous sequence determination during the polymerase reaction. Thus, real-time single-molecule electronic DNA sequencing data with single-base resolution were obtained. The use of these polymer-tagged nucleotides, combined with polymerase tethering to nanopores and multiplexed nanopore sensors, should lead to new high-throughput sequencing methods.

The degree of Helicobacter pylori-triggered inflammation is manipulated by preinfection host microbiota.

Helicobacter pylori infects over 3 billion people worldwide and is the primary risk factor for gastric cancer. Most individuals infected with H. pylori develop only asymptomatic gastritis; however, some develop ulcers or gastric adenocarcinoma. We demonstrate that one previously unappreciated parameter influencing H. pylori disease outcome is variation in the preinfection host microbiota. Utilizing a mouse model, we altered the microbiota by antibiotic treatment and found that these alterations resulted in significantly lowered H. pylori-triggered inflammation. Specifically, antibiotic pretreatment reduced CD4(+) T-helper cells and Ifnγ transcript levels in gastric tissue after H. pylori infection. The bacterial communities in mice with a reduced response to H. pylori displayed many differences from those in untreated mice, including significantly more cluster IV and XIVa Clostridium spp., bacteria known to influence inflammation via regulatory T cell populations. Our findings suggest that microbiota composition, perhaps Clostridium spp., contributes to the variable disease outcome of H. pylori infection by altering the recruitment of CD4(+) T cells to the gastric compartment. Our results suggest that gastric microbiota could be used as a diagnostic tool to determine which patients are at risk for developing severe disease.