A SIMPLE DNA-POLYMERASE CHAIN-REACTION METHOD TO LOCATE AND DEFINE ORIENTATION OF SPECIFIC SEQUENCES IN CLONED BACTERIAL GENOMIC FRAGMENTS

A simple DNA polymerase chain reaction (PCR) method, to rapidly locate and define the orientation of a particular sequence within a cloned b acterial genomic fragment several kilobases (kb) long, is described. T he technique is particularly useful when cloning (by DNA PCR amplifica tion) a specific sequence of a conserved gene from several micro-organ isms following an homology probing approach. The method requires two u niversal primers derived from the vector, two specific primers derived from each end of the specific sequence in inverted tail-to-tail direc tions, and a single round of PCR. In addition, PCR conditions applicab le to DNA inserts having a G + C content up to 75% (e.g. Pseudomonas a nd actinomycete genomic fragments), and allowing efficient amplificati on of DNA fragments up to 7 kb long, are described and discussed.