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.
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
Ruliang
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Xu
Shuji
S
Ogino
Va
V
Lip
Hong
H
Fang
Bai-Lin
BL
Wu
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