Provided is multiplex and asymmetric amplification of nucleic acid molecules. In particular, provided is a method for simultaneous and asymmetric amplification of one or more target nucleic acids in a sample. The method can simultaneously and asymmetrically amplify multiple target nucleic acids existing in a sample, and can simultaneously produce large number of single stranded products.

Disclosed are W-position modified cytidine nucleotides of formula (I). Provided herein are methods of chemical synthesis of AP-modified cytidine nucleoside triphosphates and their applications as well as uses of the cytidine analogues for the synthesis of modified nucleic acids. The nucleic acid molecule includes DNA, RNA or a combination of DNA/RNA. One of many applications of modified cytidine nucleotides described herein is enzyme selection, when an enzyme of interest bears an activity of an esterase, amidase, oxidoreductase, lyase, ligase or other enzymatic activity, formula (I) wherein the substituants are as defined in the appended claims.

Disclosed are W-position modified cytidine nucleotides of formula (I). Provided herein are methods of chemical synthesis of AP-modified cytidine nucleoside triphosphates and their applications as well as uses of the cytidine analogues for the synthesis of modified nucleic acids. The nucleic acid molecule includes DNA, RNA or a combination of DNA/RNA. One of many applications of modified cytidine nucleotides described herein is enzyme selection, when an enzyme of interest bears an activity of an esterase, amidase, oxidoreductase, lyase, ligase or other enzymatic activity, formula (I) wherein the substituants are as defined in the appended claims.

An example flow cell includes a substrate having a surface. The flow cell also includes a polymeric hydrogel attached to at least a portion of the substrate surface, where the polymeric hydrogel includes a dark quencher. The flow cell further includes at least one primer set attached to the polymeric hydrogel.

An example flow cell includes a substrate having a surface. The flow cell also includes a polymeric hydrogel attached to at least a portion of the substrate surface, where the polymeric hydrogel includes a dark quencher. The flow cell further includes at least one primer set attached to the polymeric hydrogel.

Provided herein is a method for sample analysis. In some embodiments, the method may involve: (a) incubating a nucleic acid sample with a terminal transferase and a cyclooctene-functionalized nucleotide to produced cyclooctene-functionalized nucleic acid molecules; (b) tethering the cyclooctene-functionalized nucleic acid molecules to a tetrazine-functionalized support via an Alder cycloaddition reaction; (c) performing at least two separate primer extension reactions using the tethered nucleic acid molecules as a template to produce multiple distinct sets of primer extension products; (d) separately analyzing the sets of primer extension products using different methods to produce multiple data sets; and (e) integrating the data sets.

Provided herein is a method for sample analysis. In some embodiments, the method may involve: (a) incubating a nucleic acid sample with a terminal transferase and a cyclooctene-functionalized nucleotide to produced cyclooctene-functionalized nucleic acid molecules; (b) tethering the cyclooctene-functionalized nucleic acid molecules to a tetrazine-functionalized support via an Alder cycloaddition reaction; (c) performing at least two separate primer extension reactions using the tethered nucleic acid molecules as a template to produce multiple distinct sets of primer extension products; (d) separately analyzing the sets of primer extension products using different methods to produce multiple data sets; and (e) integrating the data sets.

Determining the sequence of a nucleic acid typically entails performing multiple cycles of a reaction that generates a signal, depending on the identity of one or more nucleotides in the sequence. Sequencing typically is done on a plurality of copies of a template to fortify the signal and to increase accuracy. However, as the number of cycles increases, some of the copies go out of phase, increasing signal-to-noise ratio and compromising accuracy. Provided is a strategy using blocking groups and dinucleotide recognition to bring each of the copies back into phase. This improves accuracy and enables the user to increase the length of sequence reads.

Determining the sequence of a nucleic acid typically entails performing multiple cycles of a reaction that generates a signal, depending on the identity of one or more nucleotides in the sequence. Sequencing typically is done on a plurality of copies of a template to fortify the signal and to increase accuracy. However, as the number of cycles increases, some of the copies go out of phase, increasing signal-to-noise ratio and compromising accuracy. Provided is a strategy using blocking groups and dinucleotide recognition to bring each of the copies back into phase. This improves accuracy and enables the user to increase the length of sequence reads.

The present invention is related to a method for adding one or more L-nucleotides to the 3′end of a first L-nucleic acid, wherein the method comprises the step of reacting the one or more L-nucleotides with the first L-nucleic acid in the presence of a protein comprising a mutant enzymatic activity exhibiting moiety, wherein the enzymatic activity is capable of adding one or more L-nucleotides to the 3′ end of the first L-nucleic acid, wherein the mutant enzymatic activity exhibiting moiety comprises an amino acid sequence, wherein the amino acids of the amino acid sequence are D-amino acids, wherein the mutant enzymatic activity exhibiting moiety is a variant of an enzymatic activity exhibiting moiety, wherein the enzymatic activity exhibiting moiety consists of an amino acid sequence according to SEQ ID NO: 15 and wherein the amino acids of the amino acid sequence according to SEQ ID NO: 15 are D-amino acids, wherein the amino acid sequence of the mutant enzymatic activity exhibiting moiety differs from the amino acid sequence of the enzymatic activity exhibiting moiety consisting of an amino acid sequence according to SEQ ID NO: 15 at least at one amino acid position, preferably at three amino acid positions, and/or wherein the amino acid sequence of the mutant enzymatic activity exhibiting moiety is a truncated form of an amino acid sequence according to SEQ ID NO: 15, and wherein the amino acid sequence of the mutant enzymatic activity exhibiting moiety is different from an amino acid sequence according to any of SEQ ID NOs 15 to 22 and 51.