A polynucleotide sequencing method comprises (i) removing a label and a blocking moiety from a blocked, labeled nucleotide incorporated into a copy polynucleotide strand that is complementary to at least a portion of a template polynucleotide strand; and (ii) washing the removed label and blocking moiety away from the copy strand with a wash solution comprising a first buffer comprising a scavenger compound. Removing the label and blocking moieties may comprise chemically removing the moieties. The first buffer may also comprise an antioxidant and may be used in a scanning buffer used during a nucleotide detection step.

A method for generating amplicon constructs of a target sequence is disclosed, the method comprising providing a target sequence; an oligonucleotide probe, comprising a universal sequence and further comprising, at or towards its 5′ end, a target specific sequence capable of hybridising to the reverse complement of a sequence at, or flanking one of the 3′ ends of the target sequence; a universal primer, comprising at its 3′ end a sequence capable of hybridising to the universal sequence of the oligonucleotide probe and performing a Polymerase Chain Reaction (PCR).

A method for generating amplicon constructs of a target sequence is disclosed, the method comprising providing a target sequence; an oligonucleotide probe, comprising a universal sequence and further comprising, at or towards its 5′ end, a target specific sequence capable of hybridising to the reverse complement of a sequence at, or flanking one of the 3′ ends of the target sequence; a universal primer, comprising at its 3′ end a sequence capable of hybridising to the universal sequence of the oligonucleotide probe and performing a Polymerase Chain Reaction (PCR).

A method for sequencing a population of nucleic acids, which includes (a) binding the population of nucleic acids with a fractionally labeled mixture of nucleotides, thereby forming a fractionally labeled population of nucleic acids, wherein the mixture includes nucleotide cognates for a common base type in the templates, and wherein a fraction of the nucleotide cognates for the common base type in the mixture are exogenously labeled nucleotides that produce a signal that is not produced by other nucleotide cognates for the common base type in the mixture; (b) detecting the signal from the fractionally labeled population of nucleic acids; and (c) repeating (a) and (b) using a second mixture of the fractionally labeled nucleotides, wherein the fraction of the exogenously labeled nucleotides is higher in the second mixture.

A method for sequencing a population of nucleic acids, which includes (a) binding the population of nucleic acids with a fractionally labeled mixture of nucleotides, thereby forming a fractionally labeled population of nucleic acids, wherein the mixture includes nucleotide cognates for a common base type in the templates, and wherein a fraction of the nucleotide cognates for the common base type in the mixture are exogenously labeled nucleotides that produce a signal that is not produced by other nucleotide cognates for the common base type in the mixture; (b) detecting the signal from the fractionally labeled population of nucleic acids; and (c) repeating (a) and (b) using a second mixture of the fractionally labeled nucleotides, wherein the fraction of the exogenously labeled nucleotides is higher in the second mixture.

The invention is directed to methods for synthesizing a plurality of oligonucleotides in the same reaction vessel, and in some embodiments, using the synthesized oligonucleotides in an oligonucleotide-based assay in such reaction vessel. In some embodiments, methods of the invention are implemented by steps of (a) providing a plurality of different initiators attached to one or more supports, each different initiator having a terminal nucleotide with a different 3-O-blocking group; (b) for each different initiator, synthesizing a polynucleotide by repeated cycles of template-free enzymatic additions of 3′-O-blocked nucleoside triphosphates, wherein the blocking group of the 3-O-blocked nucleoside triphosphate is removable under deblocking conditions orthogonal to the deblocking conditions for removing blocking groups of the other initiators; and (c) releasing the oligonucleotides from the polynucleotides and the one or more solid supports.

The invention is directed to methods for synthesizing a plurality of oligonucleotides in the same reaction vessel, and in some embodiments, using the synthesized oligonucleotides in an oligonucleotide-based assay in such reaction vessel. In some embodiments, methods of the invention are implemented by steps of (a) providing a plurality of different initiators attached to one or more supports, each different initiator having a terminal nucleotide with a different 3-O-blocking group; (b) for each different initiator, synthesizing a polynucleotide by repeated cycles of template-free enzymatic additions of 3′-O-blocked nucleoside triphosphates, wherein the blocking group of the 3-O-blocked nucleoside triphosphate is removable under deblocking conditions orthogonal to the deblocking conditions for removing blocking groups of the other initiators; and (c) releasing the oligonucleotides from the polynucleotides and the one or more solid supports.

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.