METHODS, COMPOSITIONS AND KITS FOR A ONE-STEP DNA CLONING SYSTEM

摘要

Methods and kits for joining two or more polynucleotides to form a product polynucleotide are provided. A mixture contains a first polynucleotide comprising a selectable marker. The mixture further contains a second polynucleotide comprising a first typeIIs recognition sequence and a second typeIIs recognition sequence. The second polynucleotide is other than the first polynucleotide. The mixture further contains a first typeIIs restriction endonuclease that cleaves the first typeIIs recognition sequence to produce a first end, a second typeIIs restriction endonuclease that cleaves the second typeIIs recognition sequence to produce a second end, and a DNA ligase. The first end is not compatible with the second end. The combined actions of the enzymes in the mixture join the first polynucleotide to the second polynucleotide forming a product polynucleotide, which is obtained by transforming the mixture into a host cell.

主权项

1. A method of joining two or more polynucleotides to form a product polynucleotide, the method comprising incubating a mixture for a predetermined length of time, wherein the mixture comprises:
a) a first polynucleotide comprising a selectable marker, b) a second polynucleotide, other than the first polynucleotide, comprising a first typeIIs recognition sequence and a second typeIIs recognition sequence, c) a first typeIIs restriction endonuclease that recognizes the first typeIIs recognition sequence and cleaves the second polynucleotide to produce a first end, and a second typeIIs restriction endonuclease that recognizes the second typeIIs recognition sequence and cleaves the second polynucleotide to produce a second end, and wherein the first end is not complementary to the second end, and d) a DNA ligase.