Novel methods of generating a localized population of immobilized clonal amplicons on a support are provided.

Novel methods of generating a localized population of immobilized clonal amplicons on a support are provided.

A molecular state machine is implemented in a cell by designing the cell to use specific homology directed repair (“HDR”) templates for repairing double strand breaks in polynucleotides based on a current “state” of the cell. The state may be established by the presence of a molecule in the cell or by the availability of specific cut sites in the polynucleotides of the cell. Different HDR templates or different nucleases may be available for performing HDR based on the state. When the state is changed, the same signal or event will result in a different HDR template being incorporated into the existing polynucleotides of the cell. Signals that are internal or external to the cell may be used to change the state of the cell. The cell may create a log of molecular events, store binary data, or perform other synthetic biology/molecular computing functions based on state.

A molecular state machine is implemented in a cell by designing the cell to use specific homology directed repair (“HDR”) templates for repairing double strand breaks in polynucleotides based on a current “state” of the cell. The state may be established by the presence of a molecule in the cell or by the availability of specific cut sites in the polynucleotides of the cell. Different HDR templates or different nucleases may be available for performing HDR based on the state. When the state is changed, the same signal or event will result in a different HDR template being incorporated into the existing polynucleotides of the cell. Signals that are internal or external to the cell may be used to change the state of the cell. The cell may create a log of molecular events, store binary data, or perform other synthetic biology/molecular computing functions based on state.

In some embodiments, methods for ligating nucleic acid ends comprise: conducting a nucleic acid ligation reaction in the presence of at least one agent that generates a ligatable terminal 5′ phosphate group by removing an adenylate group from a terminal 5′ phosphate of a nucleic acid. In some embodiments, an aprataxin enzyme can catalyze removal of an adenylate group from a terminal 5′ phosphate of a nucleic acid. In some embodiments, methods for ligating nucleic acid ends comprise: conducting a nucleic acid ligation reaction in the presence of an aprataxin enzyme under conditions suitable for ligating nucleic acid ends.

In some embodiments, methods for ligating nucleic acid ends comprise: conducting a nucleic acid ligation reaction in the presence of at least one agent that generates a ligatable terminal 5′ phosphate group by removing an adenylate group from a terminal 5′ phosphate of a nucleic acid. In some embodiments, an aprataxin enzyme can catalyze removal of an adenylate group from a terminal 5′ phosphate of a nucleic acid. In some embodiments, methods for ligating nucleic acid ends comprise: conducting a nucleic acid ligation reaction in the presence of an aprataxin enzyme under conditions suitable for ligating nucleic acid ends.

Methods and compositions for determining the proximity of two barcoding oligonucleotides (e.g., in a single partition or adjacent on a tissue section) using a determination of the presence of a 9 bp sequence resulting from tagmentation in different nucleic acid fragments linked to different barcoding oligonucleotides is provided.

Methods and compositions for determining the proximity of two barcoding oligonucleotides (e.g., in a single partition or adjacent on a tissue section) using a determination of the presence of a 9 bp sequence resulting from tagmentation in different nucleic acid fragments linked to different barcoding oligonucleotides is provided.

This disclosure provides for methods and reagents for rapid multiplex RPA reactions and improved methods for detection of multiplex RPA reaction products. In addition, the disclosure provides new methods for eliminating carryover contamination between RPA processes.

This disclosure provides for methods and reagents for rapid multiplex RPA reactions and improved methods for detection of multiplex RPA reaction products. In addition, the disclosure provides new methods for eliminating carryover contamination between RPA processes.