Patent ID: 11920129
Assignee: NATIONAL UNIVERSITY OF SINGAPORE
Field: Biotechnology (Chemistry)
Classification: CPC C | IPC C

Claim 0:
1. A method for ligating three nucleic acid molecules comprising:
(i) providing a first nucleic acid molecule comprising a first overhang of one nucleotide in length at a first end and a second overhang of one nucleotide of in length at its other (or second) end; wherein the first overhang and the second overhang are different nucleotides and are not complementary to each other, wherein the first overhang is a 3′ C overhang and the second overhang is a 3′ T overhang, wherein the first nucleic acid molecule is a double-stranded nucleic acid molecule comprising a coding sequence strand and a substantially complementary strand, and wherein the 3′ C overhang is on the substantially complementary strand and the 3′ T overhang is on the coding sequence strand;
(ii) providing a second nucleic acid molecule capable of forming a stem-loop structure with an overhang of one nucleotide; wherein the overhang of the second nucleic acid molecule is substantially complementary to the first overhang of the first end of the first nucleic acid molecule, wherein the overhang of the second nucleic acid molecule is a 3′ G overhang; and also providing a third nucleic acid molecule capable of forming a stem-loop structure with an overhang of one nucleotide; wherein the overhang of the third nucleic acid molecule is substantially complementary to the second overhang of the second end of the first nucleic acid molecule, wherein the overhang of the third nucleic acid molecule is a 3′ A overhang; and wherein the overhang of the second nucleic acid molecule and the overhang of the third nucleic acid molecule are different nucleotides and are not complementary to each other; and
(iii) ligating the first overhang at the first end of the first nucleic acid molecule to the overhang of the second nucleic acid molecule and also the second overhang of the second end of the first nucleic acid molecule to the overhang of the third nucleic acid molecule to form a single nucleic acid molecule.