A PCR differential screening method for rapid isolation of clones from a cDNA library.
We have developed a new two-step differential screening method that allows the rapid isolation of induced or suppressed pure gene clones from a lambda phage cDNA library. This method involves a primary differential screening step and a PCR differential screening step. From the primary screening step, impure pools of positive cDNA clones are obtained. Each pool of clones is then amplified directly by PCR using two primers flanking the cloning site in the vector. The PCR products are run on two duplicate agarose gels and blotted onto two filter strips. These two filters are then subjected to differential Southern hybridization with different cDNA probes. Each pure positive cDNA band on the gel is identified and selected for further subcloning. This method has three advantages over the traditional differential screening method. First, several rounds of plaque rescreening are replaced by a single PCR screening. Second, plaque hybridization is replaced by a more reliable and accurate PCR DNA Southern hybridization. Third, the time-consuming and tedious phage DNA isolation step is eliminated, and subcloning is facilitated by direct cloning of the pure PCR product to a plasmid vector. We have successfully used this method to isolate ozone-responsive genes from a cDNA library of monkey respiratory airways.
University of California at Davis.
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D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D015139:Blotting, Southern; D001980:Bronchi / Q000737:chemistry / Q000187:drug effects; D003001:Cloning, Molecular; D015342:DNA Probes; D018076:DNA, Complementary / Q000302:isolation & purification*; D008253:Macaca mulatta; D009693:Nucleic Acid Hybridization; D010126:Ozone / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D014132:Trachea / Q000737:chemistry / Q000187:drug effects
D015342:DNA Probes; D018076:DNA, Complementary; D010126:Ozone