The invention relates to new methods of attaching one or more polynucleotide binding proteins to a target polynucleotide. The invention also related to new methods of characterising target polynucleotides.

The disclosure provides methods, compositions, systems, and kits for the concurrent detection and analysis of different structural and chemical forms of nucleic acids in a sample.

Crosslinked nucleotide polymers. A crosslinked nucleotide polymer may be formed by reaction of a biomass comprising DNA and/or RNA with one or more crosslinker(s). A crosslinked nucleotide polymers may be formed by a crosslinking reaction including an aza-Michael addition reaction. Crosslinked nucleotide polymers may be present in various forms and compositions and form various articles of manufacture. Crosslinked nucleotide polymers may be used in therapeutic methods, coating methods, and cell-free protein production methods.

The invention provides methods of synthesizing a product DNA molecule having a desired and/or defined sequence. The methods involve annealing at least one long oligonucleotide and at least one short oligonucleotide to at least one anchor strand having a sequence at least partially complementary to the at least one long and at least one short oligonucleotide. After annealing, at least one long oligonucleotide bound to an anchor strand abuts at least one short oligonucleotide bound to the same anchor strand. The anchor strand has one or more non-standard nucleotides, and optionally one or more degenerate nucleotides. The method involves ligating the abutting at least one long oligonucleotide and at least one short oligonucleotide to form a dsDNA molecule. The invention also provides methods of synthesizing DNA molecules by assembling oligonucleotide members of a library that contains less than 20,000 members that can be assembled into all possible DNA sequences.

A number of T4 DNA ligase mutants exhibiting enhanced ligation activity at temperatures above 37° C. compared to the wild-type ligase were engineered, characterized, and selected via gel electrophoresis of ligation products from a standard ligation assay. T4 Ligase catalyzes the formation of phosphodiester bonds between the 5′ and 3′ ends of complementary cohesive ends or blunt ends of duplex DNA, a process that is vital to numerous molecular biology processes including cloning and sequencing.

Provided herein are methods of identifying a location of an RNA in a sample that include: (a) contacting the sample with an array comprising capture probes, where a capture probe comprises a capture domain and a spatial barcode; (b) releasing the RNA from the sample; (c) extending a 3′ end of the capture probe using the capture domain-bound RNA as a template; (d) generating nick(s) in the extended capture probe-hybridized RNA and performing random-primed DNA synthesis; (e) performing end repair on the second strand DNA molecule; (f) adding a single adenosine nucleotide to the 3′ end of the extended capture probe; (g) ligating a double-stranded sequencing adaptor to the double-stranded DNA product; and (h) determining all or a part of the sequence of the RNA, and the sequence of the spatial barcode, or complements thereof, and using the determined sequences to identify the location of the RNA in the sample.

Provided are a method for constructing a bispecific antigen binding polypeptide expression vector and the bispecific antigen binding polypeptide expression vector produced according to the method. The method comprises: performing treatment by using restriction endonuclease of a specific recognition enzyme cutting site to obtain seven nucleic acid fragments having specific cohesive ends, and directionally ligating the nucleic acid fragments. Further provided is a method for establishing a polypeptide display library by using the expression vector. The display library can be used for effectively screening antibodies or antibody fragments.

Provided herein are methods of identifying a location of an RNA in a sample that include: (a) contacting the sample with an array comprising capture probes, where a capture probe comprises a capture domain and a spatial barcode; (b) releasing the RNA from the sample; (c) extending a 3′ end of the capture probe using the capture domain-bound RNA as a template; (d) generating nick(s) in the extended capture probe-hybridized RNA and performing random-primed DNA synthesis; (e) performing end repair on the second strand DNA molecule; (f) adding a single adenosine nucleotide to the 3′ end of the extended capture probe; (g) ligating a double-stranded sequencing adaptor to the double-stranded DNA product; and (h) determining all or a part of the sequence of the RNA, and the sequence of the spatial barcode, or complements thereof, and using the determined sequences to identify the location of the RNA in the sample.

The present disclosure describes a method of adapter ligation to the ends of fragmented double-stranded DNA molecules.

The disclosure provides methods, compositions, systems, and kits for the concurrent detection and analysis of different structural and chemical forms of nucleic acids in a sample.