The present invention discloses a ω-transaminase mutant obtained through DNA synthetic shuffling combined mutation. The ω-transaminase mutant is obtained through point mutation of a wild type ω-transaminase from Aspergillus terrus. The amino acid sequence of the wild type ω-transaminase is shown in SEQ ID NO: 1. The mutation site of the ω-transaminase mutant is any one of: (1) F115L-H210N-M150C-M280C; (2) F115L-H210N; (3) F115L-H210N-E253A-I295V; (4) I77L-F115L-E133A-H210N-N245D; (5) I77L-Q97E-F115L-L118T-E253A-G292D; (6) I77L-E133A-N245D-G292D; and (7) H210N-N245D-E253A-G292D. According to the present invention, forward mutations obtained in the previous stage are randomly combined through a DNA synthetic shuffling combined mutation method. It is verified through experiments that this method can effectively improve the probability of forward mutation and increase experimental efficiency and feasibility, and is capable of obtaining mutant enzymes with thermodynamic stability remarkably superior to that of wild enzymes via screening.

The present invention provides methods and kits for efficiently generating edited DNA, ranging from DNA molecules with simple point mutations to complex DNA libraries. The methods and kits use a single-stranded deoxyuridine (dU)-containing template to align a series of DNA oligomers that contain mutations relative to the template strand, giving rise to the sequence variation in the product. The methods and kits may be used to produce DNA libraries or to generate multiple, widely spaced mutations in a target sequence.

The disclosure generally relates to compositions and methods for the production of nucleic acid molecules. In some aspects, the invention allows for the microscale generation of nucleic acid molecules, optionally followed by assembly of these nucleic acid molecules into larger molecules. In some aspects, the invention allows for efficient production of nucleic acid molecules (e.g., large nucleic acid molecules such as genomes).

A method for synthesizing a target double stranded (ds) polynucleotide by providing an oligonucleotide library within an array device that has a diversity of oligonucleotide library members, each of which has a different nucleotide sequence and is contained in a separate library containment in an aqueous solution. The library includes single stranded oligonucleotides and double stranded oligonucleotides with at least one overhang and covers at least 10,000 pairs of matching oligonucleotides. In a first step, transferring at least a first pair of matching oligonucleotides transferred from the library into a first reaction containment using a liquid handler and the matching oligonucleotides are assembled, thereby obtaining a first reaction product comprising at least one overhang. Further reaction products are then likewise obtained and are assembled in a predetermined workflow using an algorithm, thereby producing said target ds polynucleotide with an overhang, optionally followed by a finalization step to prepare blunt ends.

Methods of sequencing and assembling a nucleic acid sequence from a nucleic acid sample containing repetitive or low-information regions, which are typically difficult to sequence and/or assemble are provided. The methods of sequencing and assembling introduce mutations into the sample to increase sequence diversity between various repetitive regions present in the nucleic acid sample. This sequence diversity allows various segments to assemble independently of different, but similar sequences present in the nucleic acid sample.

Methods of sequencing and assembling a nucleic acid sequence from a nucleic acid sample containing repetitive or low-information regions, which are typically difficult to sequence and/or assemble are provided. The methods of sequencing and assembling introduce mutations into the sample to increase sequence diversity between various repetitive regions present in the nucleic acid sample. This sequence diversity allows various segments to assemble independently of different, but similar sequences present in the nucleic acid sample.

A library of double stranded (ds) polynucleotide library members of at least 12 bp length comprising a variety of polynucleotide core sequences and the same overhangs.

The current inventive technology includes systems, methods, and compositions for directly synthesizing sticky ended DNA fragments for subsequent gene assembly. In a preferred embodiment, the inventive technology includes strategies for the direct synthesis of sticky ended DNA with 5′ overhangs that have any desired length and base composition, using typical PCR protocols with no additional manipulation. In another embodiment, the inventive technology includes the direct synthesis of sticky ended DNA using chemically modified oligonucleotide primers in a polymerase chain reaction (PCR). In certain embodiments, the inventive technology allows for the generation of larger DNA constructs formed by the sticky-ended assemblies generally described herein compared to traditional synthesis and ligation applications.

The current inventive technology includes systems, methods, and compositions for directly synthesizing sticky ended DNA fragments for subsequent gene assembly. In a preferred embodiment, the inventive technology includes strategies for the direct synthesis of sticky ended DNA with 5′ overhangs that have any desired length and base composition, using typical PCR protocols with no additional manipulation. In another embodiment, the inventive technology includes the direct synthesis of sticky ended DNA using chemically modified oligonucleotide primers in a polymerase chain reaction (PCR). In certain embodiments, the inventive technology allows for the generation of larger DNA constructs formed by the sticky-ended assemblies generally described herein compared to traditional synthesis and ligation applications.

High-fidelity, high-throughput nucleic acid sequencing enables healthcare practitioners and patients to gain insight into genetic variants and potential health risks. However, previous methods of nucleic acid sequencing often introduce sequencing errors (for example, mutations that arise during the preparation of a nucleic acid library, during amplification, or sequencing). Provided herein are methods and compositions for sequencing nucleic acids. Further provided are methods of identifying an error in a nucleic acid sequence.