Embodiments of the present disclosure relate to nucleotides labeled with photoswitchable compounds. Also provided herein are methods and kits of using these labeled nucleotides for sequencing applications.

Provided herein are methods and compositions for creating a sequencing library comprising a target nucleic acid. Methods herein can comprise: contacting a nucleic acid sample to a first population of primers, a polymerase, dNTPs, and labeled ddNTPs; performing an extension reaction thereby creating an labeled extension product; contacting the extension product to a second population of primers to create a double stranded extension product comprising the target nucleic acid; contacting the double stranded extension product to a target specific enzyme under conditions allowing cleavage of at least a subset of the double stranded extension product thereby creating a cleaved target nucleic acid; and isolating the cleaved target nucleic acid.

Provided herein are methods and compositions for creating a sequencing library comprising a target nucleic acid. Methods herein can comprise: contacting a nucleic acid sample to a first population of primers, a polymerase, dNTPs, and labeled ddNTPs; performing an extension reaction thereby creating an labeled extension product; contacting the extension product to a second population of primers to create a double stranded extension product comprising the target nucleic acid; contacting the double stranded extension product to a target specific enzyme under conditions allowing cleavage of at least a subset of the double stranded extension product thereby creating a cleaved target nucleic acid; and isolating the cleaved target nucleic acid.

A method for detecting different analytes includes mixing different analytes with sensing probes, wherein at least some of the sensing probes are specific to respective ones of the analytes. The analytes respectively are captured by the sensing probes that are specific to those analytes. Fluorophores respectively are coupled to sensing probes that captured respective analytes. The sensing probes are mixed with beads, wherein the beads are specific to respective ones of the sensing probes, and wherein the beads include different codes identifying the analytes to which those sensing probes are specific. The sensing probes respectively are coupled to beads that are specific to those sensing probes. The beads are identified that are coupled to the sensing probes that captured analytes using at least fluorescence from the fluorophores coupled to those sensing probes. The analytes that are captured are identified.

A method for detecting different analytes includes mixing different analytes with sensing probes, wherein at least some of the sensing probes are specific to respective ones of the analytes. The analytes respectively are captured by the sensing probes that are specific to those analytes. Fluorophores respectively are coupled to sensing probes that captured respective analytes. The sensing probes are mixed with beads, wherein the beads are specific to respective ones of the sensing probes, and wherein the beads include different codes identifying the analytes to which those sensing probes are specific. The sensing probes respectively are coupled to beads that are specific to those sensing probes. The beads are identified that are coupled to the sensing probes that captured analytes using at least fluorescence from the fluorophores coupled to those sensing probes. The analytes that are captured are identified.

A method for preparing nucleic acid sequences using an enzyme, including: (1) providing a reaction substrate having a pretreated surface. (2) Disposing a nucleotide having a terminal protecting group on the pretreated surface by a reaction enzyme, and a reaction temperature is 45° C.-105° C. (3) Removing the terminal protecting group of the nucleotide by irradiation or heating. (4) Coupling another nucleotide having the terminal protecting group to the nucleotide by the reaction enzyme, and a reaction temperature is 45° C.-105° C. (5) Determining whether nucleic acid sequence is completed, and if so, obtaining the nucleic acid sequence, if otherwise repeating steps (3) and (4). The method for preparing nucleic acid sequences using an enzyme of the invention may increase the efficiency of preparing nucleic acid sequences.

A method for preparing nucleic acid sequences using an enzyme, including: (1) providing a reaction substrate having a pretreated surface. (2) Disposing a nucleotide having a terminal protecting group on the pretreated surface by a reaction enzyme, and a reaction temperature is 45° C.-105° C. (3) Removing the terminal protecting group of the nucleotide by irradiation or heating. (4) Coupling another nucleotide having the terminal protecting group to the nucleotide by the reaction enzyme, and a reaction temperature is 45° C.-105° C. (5) Determining whether nucleic acid sequence is completed, and if so, obtaining the nucleic acid sequence, if otherwise repeating steps (3) and (4). The method for preparing nucleic acid sequences using an enzyme of the invention may increase the efficiency of preparing nucleic acid sequences.

Provided herein are compositions and methods using mutant Phi29 polymerases for nucleic acid amplification. Further provided herein are methods for accurate and scalable Primary Template-Directed Amplification (PTA) nucleic acid amplification and sequencing methods, and their applications for mutational analysis in research, diagnostics, and treatment using mutant Phi29 polymerases.

Provided herein are compositions and methods using mutant Phi29 polymerases for nucleic acid amplification. Further provided herein are methods for accurate and scalable Primary Template-Directed Amplification (PTA) nucleic acid amplification and sequencing methods, and their applications for mutational analysis in research, diagnostics, and treatment using mutant Phi29 polymerases.

A polynucleotide sequencing method includes a wash step that employs a composition including a polymerase. The composition may also include a plurality of nucleotides. The composition may be configured to prevent the polymerase from incorporating one of the plurality of nucleotides into a copy polynucleotide strand. The composition may be substantially free of Mg.sup.2+.