Rapid synthesis of DNA deletion constructs for mRNA quantitation: analysis of astrocyte mRNAs

A rapid method for the synthesis of DNA fragments for competitive PCR analysis is described. This procedure takes advantage of the fact that if PCR is carried out with a mixture of re-ligated restriction digestion fragments, only those fragments containing binding sites for both PCR primers will be amplified. Following electrophoresis of the amplified mixture, the DNA fragment of desired size can be excised from an agarose gel, reamplified, and used for subsequent competitive PCR. We have used this procedure to synthesize deletion constructs for the rat glial fibrillary acidic protein (GFAP) gene, and have used competitive PCR to determine the levels of this mRNA in primary cultures of rat brain astrocytes.