A Single-Tube Nested PCR Method for SMN1 Deletion Detection in Spinal Muscular Atrophy

Figures

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  Figure 3. STNPCR assay using DBS samples from control individuals. (A) Schematic diagram of STNPCR. (B,C) Alignment of the corresponding SMN1 and SMN2 target regions analyzed in this study. Uppercase letters indicate exon sequence, whereas lowercase letters indicate flanking intronic sequence. The sequences were annotated with reference to Ensembl gene models on the human genome assembly GRCh38. SMN1 and SMN2 correspond to Ensembl gene IDs ENSG00000172062 and ENSG00000205571, respectively. The single-nucleotide difference within the exonic region corresponds to the known SMN1/SMN2 sequence distinction at c.840. Sequences highlighted in yellow are forward primers (R111 and mCOP-F), and sequences highlighted in blue are reverse primers (intron 7-R). Red letters represent different nucleotides in the SMN1 and SMN2 genes. The mCOP-F primer is an SMN1-specific primer, annealing with C nucleotide at c.840 in SMN1 exon 7, but not with T nucleotide at c.840in SMN2 exon 7. The underlines indicate the positions of the primers. (D) Gel electrophoresis data of STNPCR products. DBS samples were collected from control subjects. In this experiment, an STNPCR were performed with 20 cycles for the first…

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  Figure 4. Cycle numbers of the second stage of DBS samples. (A) Control individuals. (B) SMA patients. Second-stage cycles of 15–19 showed two bands of the primary amplicon and nested amplicon (SMN1-specific products) in the control DBS sample, but a single band of the primary amplicon in the SMA DBS sample. However, 13 cycles of the second stage in the control DBS showed a very faint band (almost undetectable) of nested amplicon (SMN1-specific product band), which may lead to a false-positive result. Mk denotes molecular markers.

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  Figure 1. Positioning and workflow of single-tube nested PCR (STNPCR) for SMA diagnosis. (A) Position of the STNPCR system among diagnostic methods for SMA. (B) Outline of the diagnostic workflow for SMA using STNPCR. Other abbreviations than STNPCR are as follows: PCR-RFLP: PCR-restriction fragment-length polymorphism, qPCR: quantitative PCR, ddPCR: droplet digital PCR, HRM: high-resolution melting analysis, MLPA: Multiplex Ligation-dependent Probe Amplification.

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  Figure 2. Schematic representation of the PCR cycling conditions of the two-stage STNPCR. The program included an initial denaturation step, a first amplification stage at 55 °C, a second amplification stage at 37 °C, an additional extension step, and a final hold at 4 °C. The red dotted line indicates the timing when the first-stage PCR ends and the second-stage PCR begins.

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  Figure 5. P/A assay of SMN1 using conventional PCR. (A) Control individuals. (B) SMA patients. The results were as expected based on RFLP analysis. However, a clear upper band was not observed in some DBS samples (C1). Although SMN1-specific products were clearly shown in the C1 sample, this sample needs to be re-examined to test the possibility of false results. The C1 sample was confirmed to be from control samples. The variation in the band intensity likely reflects differences in total SMN gene copy number or template quantity in the DBS sample.