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 bacterial genomic fragment several kilobases (kb) long, is described. The technique is particularly useful when cloning (by DNA PCR amplification) a specific sequence of a conserved gene from several microorganisms following an homology probing approach. The method requires two universal primers derived from the vector, two specific primers derived from each end of the specific sequence in inverted tail-to-tail directions, and a single round of PCR. In addition, PCR conditions applicable to DNA inserts having a G + C content up to 75% (e.g. Pseudomonas and actinomycete genomic fragments), and allowing efficient amplification of DNA fragments up to 7 kb long, are described and discussed.
Skinner
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Bioprocess Research, Central Research Division, Pfizer Inc., Groton, Connecticut 06340.
Denoya
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D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*
D004269:DNA, Bacterial