Isolating candidate inserted fragment from positive fused phage clones using quantitative PCR

Objective To isolate quickly and exactly the specific inserted fragment from fused phage clones which were obtained from cDNA library by hybridization. Methods According to the amplification principle of quantitative polymerase chain reaction (PCR) and based on the difference of original template quantity, target cDNA fragment was isolated and identified by two PCRs. Results A positive clone with specific cDNA fragment of HumGT- H1 gene was obtained from a two-phage fused clone by using this method, and the inserted fragment was verified to be the 5'cDNA sequence of HumGT-H1, 1. 9kb in length. So another hybridization screening is not necessary. Conclusion The method presented is effective and rapid in gene cloning and can greatly save time and materials.