Improved method for the allelic definition of C4A and C4B polymorphism (HLA class III)

The polymorphism of the fourth component of human serum complement (C4) is well established at the proteinic level; at the DNA level in the analysis o f C4A and C4B gene polymorphism, the PCR technique is not widely and routin ely used because it is time consuming and still presents reproducibility pr oblems. This is a serious problem because only PCR genotyping allows the es tablishment of Rodgers-Chido reverse antigenicity without the need for clas sical family segregation studies, whose samples are not always easy to obta in. The most commonly used protocol requires an initial PCR followed by nes ted amplification of all the products supposed positive or negative. The tw o reactions are set up using differing cycling conditions, primers, and mag nesium chloride concentrations. We developed a simplified procedure to easi ly obtain reproducible results and used a single protocol for all reactions . Nested PCR is made using only the positive samples, so we decrease the nu mber of samples to handle, the time spent for the work, and the reagents us ed for the reactions. Moreover, we increased the reproducibility for the ex periments.