Linear dna with enhanced resistance against exonucleases and methods for the production thereof

A method of producing a closed linear DNA product, wherein the method comprises the steps of:

• (a) digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, a truncated protelomerase sequence at a first end and a truncated protelomerase sequence at a second end, wherein the truncated protelomerase target sequences are 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 wherein the second adaptor molecule comprises a truncated protelomerase target sequence that forms a second functional protelomerase target sequence with the truncated protelomerase sequence at the second end of the digested double-stranded DNA molecule;
• and
• (c) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed linear DNA product, wherein the protelomerase closes the first and second ends of the precursor double-stranded DNA molecule at the first and second functional protelomerase target sequences.

A method of producing a closed linear DNA product, wherein the method comprises the steps of:

• (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, a truncated protelomerase sequence at a first end and a truncated protelomerase sequence at a second end, wherein the truncated protelomerase target sequences are 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 wherein the second adaptor molecule comprises a truncated protelomerase target sequence that forms a second functional protelomerase target sequence with the truncated protelomerase sequence at the second end of the digested double-stranded DNA molecule;
• and
• (c) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed linear DNA product, wherein the protelomerase closes the first and second ends of the precursor double-stranded DNA molecule at the first and second functional protelomerase target sequences.

A method of producing a partially closed linear DNA product, wherein the method comprises the steps of:

• (a) digesting a double-stranded DNA molecule with an endonuclease that cleaves a 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, and wherein the truncated protelomerase 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, wherein the truncated protelomerase target sequence of the first adaptor is non-functional and wherein the second adaptor molecule is a nucleic acid molecule that comprises one or more nucleotide resistant nucleotides;
• and
• (c) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the partially closed linear DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequence.

A method of producing a closed linear DNA product, wherein the method comprises the steps of:

• (a) digesting a double-stranded DNA molecule with an endonuclease that cleaves a 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, and wherein the truncated protelomerase 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 closed linear DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequence, and the second end is closed by the second adaptor molecule.