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 SMN1 gene have been widely used in genetic testing for spinal muscular atrophy (SMA). We compared the most commonly used PCRrestriction 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 SMN1 from SMN2 at the 100-pg DNA level (DraIdigested SMN2 fragments served as an internal control for PCR efficiency). In contrast, allele-specific PCR for SMN1, 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 SMN1 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 allelespecific PCR may be considered with caution under certain circumstances.
Xu
Ruliang
R
Department of Pathology, Harvard Medical School, and Department of Pathology, Brigham and Women's Hospital, Boston, MA 02115, USA. bai-lin.wu@tch.harvard.edu
Ogino
Shuji
S
Lip
Va
V
Fang
Hong
H
Wu
Bai-Lin
BL
D000483:Alleles; D017362:Cyclic AMP Response Element-Binding Protein; D005091:Exons; D005820:Genetic Testing / Q000379:methods; D006801:Humans; D009134:Muscular Atrophy, Spinal / Q000175:diagnosis / Q000235:genetics*; D009419:Nerve Tissue Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D019836:Preimplantation Diagnosis; D016601:RNA-Binding Proteins; D055532:SMN Complex Proteins; D012680:Sensitivity and Specificity; D055533:Survival of Motor Neuron 1 Protein; D055540:Survival of Motor Neuron 2 Protein
D017362:Cyclic AMP Response Element-Binding Protein; D009419:Nerve Tissue Proteins; D016601:RNA-Binding Proteins; D055532:SMN Complex Proteins; C526471:SMN1 protein, human; C526477:SMN2 protein, human; D055533:Survival of Motor Neuron 1 Protein; D055540:Survival of Motor Neuron 2 Protein