Diagnosis of mutations by the PCR double RFLP method (PCR-dRFLP).

The diagnosis of mutations that are responsible for human genetic diseases is important, and several molecular biology techniques based on polymerase chain reaction (PCR) (1), have been specially developed for this purpose (2-8) . For some of these techniques it is the presence or the absence of a PCR product that constitutes the analytical step, ie proves the presence of the mutation. In order to discriminate between wild-type and mutant alleles, the analytical PCR requires specific and adapted PCR conditions for each mutation studied (concentration of each PCR primers set, temperature of hybridization, concentration of MgClJ. Consequently, conditions must be continually adapted for the study of several different mutations. However when a mutation creates or destroys a natural or an artificial (5 — 8) restriction site then PCR-RFLP is usually used to diagnose the mutation despite all other available diagnostic techniques. However PCR-RFLP presents a technical difficulty since a defective restriction enzyme activity can be confused with the loss of the restriction site. Thus, it has been recommended that PCR products are digested in the presence of an excess of restriction enzyme for a long period of time (8). Despite these precautions, the presence of a restriction site is experimentally easier to ascertain than its absence. In order to overcome this difficulty, we have developed a method which we call the PCR double RFLP (PCR-dRFLP). For each studied DNA, two modified nested PCRs are performed at the same time, one pair of PCR primers is designed to introduce a restriction site specific for the wild-type allele while the second pair of primers is designed to introduce a restriction site specific for the mutant allele. Each PCR product is then analyzed by RFLP. These two RFLP allow an unambiguous interpretation of the results. Essentially, it is as though each mutation abolished a restriction site on the wild-type sequence to create a new one on the mutant sequence. Furthermore, the PCR-dRFLP was developed in a manner which allows all studied mutations to be processed with a unique procedure.