Disclosed are new and improved methods and systems for nucleic acid sequence analysis that can analyze data indicative of natural by-products of nucleotide incorporation events without the need for exogenous labels or dyes to identify nucleic acid sequences of interest. In particular, the methods and systems of the present teachings can process such data and various forms thereof to align fragments of the nucleic acid(s) of interest, particularly those analyzed using an addition sequencing technique, for example, as occurs with the use of nucleotide flows.

A buffered suspension includes a surfactant and a solid buffer particulate having a point of zero charge at least 1.2 pH units different that the pH of the buffered suspension. The buffered suspension can be prepared by mixing a stock solution with the solid buffer particulate and titrating. A method of preforming a pH sensitive process includes drawing the buffered suspension from a reservoir, filtering the solid buffer particulate from the buffered suspension, and applying the filtered solution to a sensor.

A buffered suspension includes a surfactant and a solid buffer particulate having a point of zero charge at least 1.2 pH units different that the pH of the buffered suspension. The buffered suspension can be prepared by mixing a stock solution with the solid buffer particulate and titrating. A method of preforming a pH sensitive process includes drawing the buffered suspension from a reservoir, filtering the solid buffer particulate from the buffered suspension, and applying the filtered solution to a sensor.

A method and an apparatus for nucleic acid sequencing are provided. The method includes immobilizing capturing oligonucleotides with different sequences in reaction wells, immobilizing single-stranded nucleic acid templates in the reaction wells via annealing between the templates and the capturing oligonucleotides, amplifying the immobilized nucleic acid templates and producing a population of template clones annealed with a plurality of sequencing primers. The method further includes sequentially disposing different types of nucleotide trisphosphates, detecting, by ion-sensitive field-effect transistors, ion concentration change in the reaction wells in response to incorporation of one of the nucleotide trisphosphates at 3′ end of sequencing primers, when the nucleotide trisphosphates is complementary to a corresponding nucleotide in the template clones, and sequencing the template clones by repeating the sequentially disposing and the detecting. A method for producing single-stranded nucleic acid template clones on a reaction well array is also provided.

A method and an apparatus for nucleic acid sequencing are provided. The method includes immobilizing capturing oligonucleotides with different sequences in reaction wells, immobilizing single-stranded nucleic acid templates in the reaction wells via annealing between the templates and the capturing oligonucleotides, amplifying the immobilized nucleic acid templates and producing a population of template clones annealed with a plurality of sequencing primers. The method further includes sequentially disposing different types of nucleotide trisphosphates, detecting, by ion-sensitive field-effect transistors, ion concentration change in the reaction wells in response to incorporation of one of the nucleotide trisphosphates at 3′ end of sequencing primers, when the nucleotide trisphosphates is complementary to a corresponding nucleotide in the template clones, and sequencing the template clones by repeating the sequentially disposing and the detecting. A method for producing single-stranded nucleic acid template clones on a reaction well array is also provided.

A method of detecting a ligase expressing micro-organism in a sample comprises steps of treating the sample under conditions that inhibit the activity of ATP-dependent ligase from mammalian cells but which do not inhibit the activity of the microbial ligases, contacting the sample or a portion of the sample with a nucleic acid molecule which acts as a substrate for ligase activity in the sample, incubating the thus contacted sample under conditions suitable for ligase activity; and specifically determining the presence and/or the amount of a ligated nucleic acid molecule resulting from the action of the ligase on the substrate nucleic acid molecule to indicate the presence of the ligase expressing micro-organism. The micro-organism may be a fungus or a bacterium or both. High pH conditions may be employed to inactivate mammalian ligases. Related kits are described.

Techniques, systems, and devices are disclosed for non-thermal cycling of polymerase chain reaction (PCR). In one aspect, a method for cycling PCR includes receiving an electrolytic fluid including ions, primers, polymerase enzymes, nucleotides, and a double-stranded nucleic acid in a fluid chamber having a first electrode and a second electrode, applying an electric field across the first and the second electrodes to generate a first pH level of the electrolytic fluid to denature the double-stranded nucleic acid to at least partial single strands, and applying a second electric field across the first and second electrodes to produce a second pH level of the electrolytic fluid, in which the second pH level enables binding of a polymerase enzyme and a primer with a corresponding segment of the single strands.

Techniques, systems, and devices are disclosed for non-thermal cycling of polymerase chain reaction (PCR). In one aspect, a method for cycling PCR includes receiving an electrolytic fluid including ions, primers, polymerase enzymes, nucleotides, and a double-stranded nucleic acid in a fluid chamber having a first electrode and a second electrode, applying an electric field across the first and the second electrodes to generate a first pH level of the electrolytic fluid to denature the double-stranded nucleic acid to at least partial single strands, and applying a second electric field across the first and second electrodes to produce a second pH level of the electrolytic fluid, in which the second pH level enables binding of a polymerase enzyme and a primer with a corresponding segment of the single strands.

This disclosure is directed, in part, toward preparing and utilizing tunable electrostatic capture (“TEC”) ligands that have an ionizable function. The electrostatic nature of the ionizable functionality of the TEC ligands can be “tuned” or adjusted to either reversibly bind or release a desired target anion, such as a biomolecule, by varying the pH and/or the ionic strength of the binding conditions and release conditions. The TEC ligands can be bound to a solid support to form TEC binding surfaces. TEC surfaces, ligands, solid supports and the accompanying methods and buffer systems can be used to isolate polyanions, such as nucleic acids, from materials, for example in a size-selective manner.

This disclosure is directed, in part, toward preparing and utilizing tunable electrostatic capture (“TEC”) ligands that have an ionizable function. The electrostatic nature of the ionizable functionality of the TEC ligands can be “tuned” or adjusted to either reversibly bind or release a desired target anion, such as a biomolecule, by varying the pH and/or the ionic strength of the binding conditions and release conditions. The TEC ligands can be bound to a solid support to form TEC binding surfaces. TEC surfaces, ligands, solid supports and the accompanying methods and buffer systems can be used to isolate polyanions, such as nucleic acids, from materials, for example in a size-selective manner.