Patent ID: 6730500
Filing Date: 2004-05-04
Classification: C12N

Abstract:
A method for producing a nucleic acid molecule that comprises a continuous nucleotide sequence capable of being transcribed and translated to produce a protein of interest, the nucleic acid molecule derived from noncontiguous nucleotide sequences, comprising:(a) amplifying at least two nucleotide sequences from a single nucleic acid molecule template using primer pairs to produce double-stranded amplified products, wherein the amplified nucleotide sequences reside noncontiguously in the nucleic acid molecule template, wherein each primer of a primer pair comprises an uninterrupted recognition sequence for a class IIS restriction endonucicase which is located near the 5â€²-end of the primer and a portion of an amino acid coding sequence, such that cleavage of the amplified products with the class IIS restriction endonuclease yields at least two nucleic acid molecule fragments with cohesive ends that, when ligated to each other, produce a continuous nucleotide sequence capable of being transcribed and translated to produce a protein of interest, (b) cleaving amplified products with the class IIS restriction endonuclease to produce nucleic acid molecule fragments, and (c) ligating cleaved nucleic acid molecule fragments to produce a nucleic acid molecule comprising the continuous nucleotide sequence capable of being transcribed and translated to produce a protein of interest.