In contrast with the scanning detection methods (RNase A, DGGE, SSCP) that cannot characterize the mutations, the PCR/RFLP approach combines detection and characterization capabilities. The generation or destruction of restriction sites allows the rapid detection of point mutations after the genomic sequences are amplified by the PCR. In the former case, the mutation is cleaved by the specific restriction endonuclease, while the wild-type sequence is not. Gel electrophoresis easily identifies the mutations, since they generate smaller DNA fragments [137]. The situation is reversed when the mutation destroys the restriction site previously present in the wild-type sequence. In this case the mutations are evidenced by the presence of undigested DNA fragments [138, 139].
Although many point mutations do not create or abolish a restriction site and
therefore cannot be detected by this straightforward approach, the use of PCR
primers containing mismatches relative to the target sequences can circumvent
this limitation. The DNA fragments incorporate the sequence of the primers that
can be designed to create a new RFLP [137, 139, 140].
The main advantage of this approach is that it does not require the use of
radioactive isotopes and it is more amenable therefore to analyses in clinical
settings.
Adrenergic Receptor Codon