Patent ID: 6410220
Filing Date: 2002-06-25
Classification: C12N

Abstract:
A method for isolating and identifying promoters comprising the steps of:a) providing a vector comprising a portion of a promoter region from a retrovirus transposon Long Terminal Repeat (LTR) and having two non-complementary overhanging termini, said vector being generated by digesting the vector with one or more class IIS restriction enzymes, creating non-complementary termini for directional joining of promoter sequences with the vector; b) designing two PCR primers to amplify a region of a retrovirus transposon LTR promoter from template nucleic acid to produce a nucleic acid fragment, wherein said primers comprise at least one predetermined restriction endonuclease recognition site that recognizes a restriction endonuclease that cleaves at a distance from the recognition site and creates non-palindromic overhanging termini, a sequence complementary to the template nucleic acid, and bases positioned at the restriction endonuclease cleavage site that form non-palindromic overhanging termini after cleavage by the restriction endonuclease and are selected to be complementary to only one of the overhanging termini of the vector; c) combining the primers with template nucleic acid and performing a polymerase chain reaction to produce multiple copies of the template nucleic acid to form amplified template fragments incorporating the restriction endonuclease recognition site; d) digesting the amplified template fragments with one or more restriction endonucleases that recognize the restriction endonuclease recognition site of the primers to create digested template fragments having non-palindromic overhanging termini that are complementary to the respective termini of the vector; e) combining the digested template fragments in a ligation reaction with the vector to produce a gene vector with an intact LTR sequence; f) transforming the vector with an intact LTR sequence into at least one cell type; and g) selecting for expression from the LTR sequence in said cell type; whereby expression from the LTR sequence identifies a promoter.