RESUMO
Exposure of rats to peroxisome proliferators induces members of the cytochrome P450 4A (CYP4A) family. In rats, the CYP4A family consists of four related genes, CYP4A1, CYP4A2, CYP4A3, and CYP4A8. We are specifically interested in examining CYP4A1, CYP4A2, and CYP4A3, each of which is expressed in a tissue-dependent and sex-dependent manner. While CYP4A1 is sufficiently different from the other two members to enable relatively easy specific quantitation, the close similarity between CYP4A2 and CYP4A3 makes quantitative discrimination difficult. We have combined a fluorescent real-time PCR assay (TaqMan) with the sequence-specific mismatch amplification mutation assay (MAMA) to allow us to carry out specific quantitation of all three members of this family. The assay is designed such that a single fluorescent TaqMan(R) probe binds to all three gene products, while specificity is conferred by sequence-specific primers. This specific MAMA technique takes advantage of the ability of Taq polymerase to distinguish between the two cDNAs based on mismatches at the 3' end of a PCR primer. In the 84-base PCR product used for this assay, there is only a single-base difference between CYP4A2 and CYP4A3. Despite this similarity, there is at least a 1000-fold discrimination between the two sequences, using CYP4A2 or CYP4A3 specific standards. Analysis of rat liver RNA from both sexes demonstrates that this discrimination is also achieved in complex RNA mixtures. This technique should be broadly applicable to other areas of research such as allelic discrimination, detecting mutational hotspots in tumors, and discrimination among closely related members of other gene families.
