Methods for producing a linear deoxyribonucleic acid (DNA) product with enhanced resistance to nuclease digestion are provided. The methods comprise, (a) digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, and a truncated protelomerase sequence at a first end, wherein the truncated protelomerase target sequence is non-functional; (b) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule; and (c) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the linear DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequence.

Provided herein are methods, systems, and compositions for seamless nucleic acid assembly. Methods, systems, and compositions as provided herein provide for efficient assembly of nucleic acids without primer removal. Methods, systems, and compositions for seamless nucleic acid assembly comprise use of an endonuclease or exonuclease, optionally in conjunction with additional enzymes to assemble nucleic acids or polynucleotides.

Provided herein are methods, systems, and compositions for seamless nucleic acid assembly. Methods, systems, and compositions as provided herein provide for efficient assembly of nucleic acids without primer removal. Methods, systems, and compositions for seamless nucleic acid assembly comprise use of an endonuclease or exonuclease, optionally in conjunction with additional enzymes to assemble nucleic acids or polynucleotides.

The present invention provides methods, kits, and compositions for detecting a target nucleic acid. The methods comprise amplifying the target nucleic acid with a tagged primer, digesting the resulting amplicon with exonuclease III, hybridizing a tagged probe to the digested amplicon, and detecting the resulting dual-tagged amplicon-probe hybrid.

Disclosed is a DNA assembly mix, comprising a 3-5 exonuclease enzyme which is XthA; and a buffer. Also disclosed is a DNA assembly mix, comprising a polymerase and ligase free composition comprising a 3-5 exonuclease enzyme; and a buffer. Also disclosed is a method of assembling a plurality of DNA fragments, comprising: (a) mixing the plurality of DNA fragments with the DNA assembly mix as disclosed herein; and (b) incubating the mixture from step (a) at a temperature for a period of time suitable for assembling the plurality of DNA fragments. Further disclosed is use of the DNA assembly mix as disclosed herein in high-throughput DNA assembly, wherein the DNA assembly mix is used in a microfluidic platform to assemble DNA.

Sequencing adaptors and methods are provided for preparation of polynucleotides for sequencing. The sequencing adaptors contain a portion of a recognition sequence for a methyl-dependent endonuclease. Unwanted adaptor dimers that form during ligation of adaptors to target polynucleotides produce a complete restriction sequence and are cleaved by the endonuclease, followed by exonuclease digestion, thereby removing the dimers.

Provided are a method for preparing a linear closed DNA and a plasmid for use in the method. The plasmid comprises two editable regions of prokaryotic telomerase target sequences which are connected in tandem in the same direction, and both ends of the editing regions are independently provided with one or more restriction endonuclease digestion sites. The method can yield high-purity LcDNA and can be better applied to clinical research and commercial applications.