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[Construction of a competitor for bcr-abl cDNA by recombinant PCR]
1Department of Medical Genetics, Sun Yat-sen University of Medical Sciences, Guangzhou, Guangdong Province, 510089 P.R.China.
Objective:
Competitive PCR is a powerful method for quantification of mRNA. This study sought to construct an internal standard which can be used as a competitor for bcr-abl fusion cDNA.
Methods:
Using recombinant PCR for site-directed mutagenesis. RESULTS A 240bp mimic of a 276bp fragment in b3a2 type of bcr-abl cDNA was obtained. The difference in mimic,which was verified by DNA sequencing, was a 55bp deletion of the target sequence and a 19bp insertion of exogenous unrelated sequence with a XbaI restriction site. In it, except for the inner 36bp, the mimic contained the same sequence and shared the same primer recognition sites as the target b3a2 cDNA(276bp) or as the target b2a2 cDNA(201bp). The obtained recombinant vector, named pGEM-mimic, can be used as a common competitor for either b3a2 or b2a2 type of bcr-abl cDNA.
Conclusion:
This provided a simple and reliable method for constructing an internal standard used in competitive PCR.