Newborn Screening for Spinal Muscular Atrophy: DNA Preparation from Dried Blood Spot and DNA Polymerase Selection in PCR

摘要

[Background] Polymerase chain reaction (PCR) analysis using DNA from dried blood spot (DBS) sampleson filter paper is a critical technique for spinal muscular atrophy (SMA) newborn screening. However,DNA extraction from DBS is time-consuming, and elimination of PCR inhibitors from DBS is almostimpossible. [Methods] Exon 7 of the two homologous SMA-related genes, survival motor neuron (SMN) 1and SMN2, of five SMA patients and five controls were amplified by PCR with a punched-out circle of theDBS paper. Two types of DNA preparation methods were tested; DNA-extraction (extracted DNA wasadded in a PCR tube) and non-DNA-extraction (a punched-out DBS circle was placed in a PCR tube). Asfor the DNA polymerases, two different enzymes were compared; TaKaRa Ex TaqTM and KOD FX NeoTM.To test the diagnostic quality of PCR products, RFLP (Restriction fragment length polymorphism)analysis with DraI digestion was performed, differentiating SMN1 and SMN2. [Results] In PCR usingextracted DNA, sufficient amplification was achieved with TaKaRa Ex TaqTM and KOD FX NeoTM, andthere was no significant difference in amplification efficiency between them. In direct PCR with apunched-out DBS circle, sufficient amplification was achieved when KOD FX NeoTM polymerase was used,while there was no amplification with TaKaRa Ex TaqTM. RFLP analysis of the direct PCR products withKOD FX NeoTM clearly separated SMN1 and SMN2 sequences and proved the presence of both of SMN1and SMN2 in controls, and only SMN2 in SMA patients, suggesting that the direct PCR products withKOD FX NeoTM were of sufficient diagnostic quality for SMA testing. [Conclusion] Direct PCR with DNApolymerases like KOD FX NeoTM has potential to be widely used in SMA newborn screening in the nearfuture as it obviates the DNA extraction process from DBS and can precisely amplify the target sequencesin spite of the presence of PCR inhibitors.