A method for one-pot one-step assembly of two or more DNA molecules to form at least one recombinant DNA molecule, and a substrate and a kit for this purpose. A simple and cost-effective assembly method for DNA molecules. A method for one-pot one-step assembly of two or more DNA molecules to form at least one recombinant DNA molecule is provided, wherein the two or more DNA molecules to be assembled are brought together in dry form with a suitable reaction medium on at least one substrate present in a reaction vessel.

Provided herein, among other things, is a conjugate comprising a polymerase and a nucleoside triphosphate, where the polymerase and the nucleoside triphosphate are covalently linked via a linker that comprises a cleavable linkage. A set of such conjugates, where the conjugates correspond to G, A, T (or U) and C is also provided. Methods for synthesizing a nucleic acid of a defined sequence are also provided. The conjugates can also be used for sequencing applications.

Provided herein, among other things, is a conjugate comprising a polymerase and a nucleoside triphosphate, where the polymerase and the nucleoside triphosphate are covalently linked via a linker that comprises a cleavable linkage. A set of such conjugates, where the conjugates correspond to G, A, T (or U) and C is also provided. Methods for synthesizing a nucleic acid of a defined sequence are also provided. The conjugates can also be used for sequencing applications.

Provided are an isolated oligonucleotide and a use thereof in nucleic acid sequencing, wherein the isolated oligonucleotide comprises a first strand, wherein the 5′-end nucleotide of the first strand has a phosphate group, and the 3′-end nucleotide of the first strand is a dideoxynucleotide, and a second strand, wherein the 5′-end nucleotide of the second strand does not have a phosphate group, and the 3′-end nucleotide of the second strand is a dideoxynucleotide, wherein the first strand is longer than the second strand in length, and a double-stranded structure is formed between the first strand and the second strand.

Provided are an isolated oligonucleotide and a use thereof in nucleic acid sequencing, wherein the isolated oligonucleotide comprises a first strand, wherein the 5′-end nucleotide of the first strand has a phosphate group, and the 3′-end nucleotide of the first strand is a dideoxynucleotide, and a second strand, wherein the 5′-end nucleotide of the second strand does not have a phosphate group, and the 3′-end nucleotide of the second strand is a dideoxynucleotide, wherein the first strand is longer than the second strand in length, and a double-stranded structure is formed between the first strand and the second strand.

Methods, compositions, kits and apparatuses that include a fluid, the fluid containing a ternary complex and Li.sup.+, wherein the ternary complex includes a primed template nucleic acid, a polymerase, and a nucleotide cognate for the next correct base for the primed template nucleic acid molecule. As an alternative or addition to Li.sup.+, the fluid can contain betaine or a metal ion that inhibits polymerase catalysis such as Ca.sup.2+. In addition to Li.sup.+, the fluid can contain polyethylenimine (PEI) with or without betaine.

Methods, compositions, kits and apparatuses that include a fluid, the fluid containing a ternary complex and Li.sup.+, wherein the ternary complex includes a primed template nucleic acid, a polymerase, and a nucleotide cognate for the next correct base for the primed template nucleic acid molecule. As an alternative or addition to Li.sup.+, the fluid can contain betaine or a metal ion that inhibits polymerase catalysis such as Ca.sup.2+. In addition to Li.sup.+, the fluid can contain polyethylenimine (PEI) with or without betaine.

Methods of detecting the absence or presence of a micro-organism in a sample comprising: contacting the sample with a nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the micro-organism in the sample, incubating the thus contacted sample under conditions suitable for nucleic acid modifying activity; and specifically determining the absence or presence of a modified nucleic acid molecule resulting from the action of the nucleic acid modifying activity on the substrate nucleic acid molecule to indicate the absence or presence of the micro-organism. Corresponding kits are also provided.

The present disclosure provides compositions, methods, kits, systems and apparatus that are useful for nucleic acid polymerization. In particular, modified polymerases and biologically active fragments thereof, such as modified Taq polymerases, are provided that allow for improved nucleic acid amplification. In some aspects, the disclosure provides modified polymerases having improved thermostability, accuracy, processivity and/or read length as compared to a referenceTaq polymerase. In some aspects, the disclosure relates to modified polymerases or biologically active fragments thereof, useful for amplification methods, and in practically illustrative embodiments, emulsion PCR.

Apparatus and methods for analyzing single molecule and performing nucleic acid sequencing. An integrated device includes multiple pixels with sample wells configured to receive a sample, which, when excited, emits radiation; at least one element for directing the emission radiation in a particular direction; and a light path along which the emission radiation travels from the sample well toward a sensor. The apparatus also includes an instrument that interfaces with the integrated device. Each sensor may detect emission radiation from a sample in a respective sample well. The instrument includes an excitation light source for exciting the sample in each sample well.