SMN1 (SMA) PCR Assay

Introduction

Spinal muscular atrophy (SMA) is a neurodegenerative disease that affects alpha-motor neurons in the spine. The prevalence of SMA is approximately 1/10.000 with a carrier frequency of 1/40 to 1/60 but may differ significantly in some ethnic groups (1/35 to 1/117). For most SMA cases, the responsible region for the disease is located on chromosome 5q11.2-13.3. The corresponding genes comprise the telomeric survival motor neuron gene 1 (SMN1) and the centromeric survival motor neuron gene 2 (SMN2). Both genes differ only by a few nucleotides. The SMN2 gene codes for the same protein as the SMN1 gene, but SMN2 transcripts are rather unstable because of the variant c.840C>T that causes exon 7 skipping in 90% of SMN2 splicing products. The presence of intact SMN2 gene copies is a modulator of SMA. However, exon 7 and 8 deletions in the SMN1 gene are causative for >95% of SMA cases. The absence of exon 7 (D7SMN1) in both SMN1 gene copies has a clinical sensitivity of 95% and a clinical specificity of >99% in the diagnostics of SMA.

Storage and Stability

SMN1 RealFast™ Assay is shipped on cooling blocks. On arrival, store the kit at -30 to -15°C. Alternatively, store at 2 to 8°C for short-term use within one month. The kit withstands up to 20 freeze/thaw cycles with no loss of activity. Avoid prolonged exposure to intense light. If stored correctly, the kit will retain full activity until the expiration date indicated on the label.

Principle of the Test

The test is based on the fluorogenic 5’ nuclease assay, also known as TaqMan® assay. Each reaction contains gene-specific primer pairs that amplify a 142 bp fragment of the SMN1 gene and a 147 bp fragment of a control gene, the latter serving as the PCR control. Further components are two dual-labeled, gene-specific hydrolysis probes that hybridize to the target sequence of the corresponding fragment. The proximity of the 5’-fluorescent reporter and 3’-quencher dye on intact probes prevents the reporter from fluorescing. During the extension phase of PCR the 5’ – 3’ exonuclease activity of the Taq DNA polymerase cleaves the 5’-fluorescent reporter from the hybridized probe. The physical separation of the fluorophore from the quencher dye generates a fluorescent signal in real time, which is proportional to the accumulated PCR product.

In samples positive for SMN1 c.840C both, the FAM-labeled SMN1 probe as well as the ROX-labeled PCR control probe bind to the respective gene fragments. A strong fluorescence signal is detected in the FAM channel (520 nm) and the ROX channel (605 nm). In samples negative for SMN1 c.840C, only the ROX-labeled PCR control probe hybridizes to the complementary strand of the control gene fragment. A strong fluorescence signal is detected in the ROX channel and no or only a baseline signal in the FAM channel.

Real-time PCR Instrument Compatibility

The SMN1 RealFast™ Assay is validated for use with the Bio-Rad CFX 96, Bio-Rad CFX Opus 96, and Applied Biosystems Quantstudio 5 real-time PCR instruments. The kit is compatible with various common real-time PCR instruments capable of recording FAM and ROX fluorescence:

• AB QuantStudio 5 (Applied Biosystems)
• CFX96™ (Bio-Rad)
• CFX Opus 96

The kit is supplied without PCR-grade water.

Assay Performance Specifications

• This prototype of the SMN1 RealFast™ Assay is for research use only.
• Analytical performance data: currently not available.
• Limit of detection: data not available.
• Recommended DNA concentration: 0.2 to 10 ng/µl genomic DNA.

• RealFast™ 4x LIT-Mix: 1 vial, white cap, 550 µl
• SMN1 Assay Mix: 1 vial, purple cap, 275 µl
• SMN1 WT-Control: 1 vial, green cap, 75 µl
• SMN1 MUT-Control: 1 vial, red cap, 75 µl

The RealFast™ 4x LIT-Mix comprises HotStart Taq DNA polymerase and dNTPs in an optimized buffer system. The SMN1 Assay Mix consists of gene-specific primers and dual-labeled hydrolysis probes for SMN1 and a control gene. Controls representing wild type (WT-Control) and homozygous mutant (MUT-Control) genotypes are supplied with the kit.

The kit contains reagents for 100 reactions in a final volume of 20 µl each.