81011913

open access

Version of Record

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

English (英語)

The Kobe journal of the medical sciences

65(3)

95-99

Kobe University School of Medicine

2019

2019-12-04

[Background] Polymerase chain reaction (PCR) analysis using DNA from dried blood spot (DBS) samples on 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 almost impossible. [Methods] Exon 7 of the two homologous SMA-related genes, survival motor neuron (SMN) 1 and SMN2, of five SMA patients and five controls were amplified by PCR with a punched-out circle of the DBS paper. Two types of DNA preparation methods were tested; DNA-extraction (extracted DNA was added in a PCR tube) and non-DNA-extraction (a punched-out DBS circle was placed in a PCR tube). As for 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 using extracted DNA, sufficient amplification was achieved with TaKaRa Ex TaqTM and KOD FX NeoTM, and there was no significant difference in amplification efficiency between them. In direct PCR with a punched-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 with KOD FX NeoTM clearly separated SMN1 and SMN2 sequences and proved the presence of both of SMN1 and SMN2 in controls, and only SMN2 in SMA patients, suggesting that the direct PCR products with KOD FX NeoTM were of sufficient diagnostic quality for SMA testing. [Conclusion] Direct PCR with DNA polymerases like KOD FX NeoTM has potential to be widely used in SMA newborn screening in the near future as it obviates the DNA extraction process from DBS and can precisely amplify the target sequences in spite of the presence of PCR inhibitors.

紀要論文