Patent ID: 10253321

Abstract:
Methods and kits for joining three or more polynucleotides to form a product polynucleotide are provided. Such a method includes forming a reaction mixture comprising (i) a vector fragment whose ends have four base overhangs resulting from cleavage of a vector with a first type IIs enzyme, (ii) a first insert nucleic acid with a four base overhang at one end resulting from cleavage by the first type IIs enzyme and a three base overhang at the other end resulting from cleavage by the second type IIs enzyme; and (iii) a second insert nucleic acid with a four base overhang at one end resulting from cleavage by the first type IIs enzyme and a three base overhang at the other end resulting from cleavage by the second type IIs enzyme, and (iv) a ligase. The four base overhangs of the vector ligate with the four base overhangs of the first and second inserts and the three base overhangs of the first and second inserts ligate with each other or three base overhangs of a spacer nucleic acid resulting from cleavage with the second type IIs enzyme to form a product polynucleotide in which the first and second insert nucleic acids are joined to the vector fragment.