Comparison of PCR-RFLP with allele-specific PCR in genetic testing for spinal muscular atrophy

PCR-based methods for the detection of homozygous deletion of exon 7 of the SMNI gene have been widely used in genetic testing for spinal muscular atrophy (SMA). We compared the most commonly used PCR-restriction fragment length polymorphism (PCR-RFLP) assay with an allele-specific PCR method, evaluating their potential application in direct testing, prenatal prediction, and preimplantation diagnosis, in terms of a range of DNA amounts used in such testing. We showed that PCR-RFLP could identify the SMN1 exon 7 by amplifying 10 pg of genomic DNA, and could differentiate SMNI from SMN2 at the 100-pg DNA level (DraI-digested SMN2 fragments served as an internal control for PCR efficiency). In contrast, allele-specific PCR for SMNI, despite some advantages in a rapid preimplantation diagnosis, quickly lost its specificity when 100 pg of genomic DNA was used. In addition, the absence of a SMNI fragment at the 10-pg DNA level may be due to a PCR amplification failure, and, thus, it is difficult to interpret without a proper internal control. Our data indicate that PCR-RFLP can be used for most diagnostic purposes, whereas the use of allele-specific PCR may be considered with caution under certain circumstances.