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[Isolating candidate inserted fragment from positive fused phage clones using quantitative PCR]
1Institute of Genetics,Fudan University, Shanghai,200433 P.R. China. Longyu@fudan.edu.cn
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.
