BACKGROUND: Spinal muscular atrophy (SMA) is a frequent autosomal recessive disorder, characterized by lower motor neuron loss in the spinal cord. More than 95% of SMA patients show homozygous survival motor neuron 1 (SMN1) deletion. We previously developed a screening system for SMN1 deletion based on a modified competitive oligonucleotide priming-PCR (mCOP-PCR) technique. However, non-specific amplification products were observed with mCOP-PCR, which might lead to erroneous interpretation of the screening results. AIM: To establish an improved version of the mCOP-PCR screening system without non-specific amplification. METHODS: DNA samples were assayed using a new version of the mCOP-PCR screening system. DNA samples had already been genotyped by PCR-restriction fragment length polymorphism (PCR-RFLP), showing the presence or absence of SMN1 exon 7. The new mCOP-PCR method contained a targeted pre-amplification step of the region, including an SMN1-specific nucleotide, prior to the mCOP-PCR step. mCOP-PCR products were electrophoresed on agarose gels. RESULTS: No non-specific amplification products were detected in electrophoresis gels with the new mCOP-PCR screening system. CONCLUSION: An additional targeted pre-amplification step eliminated non-specific amplification from mCOP-PCR screening.
BACKGROUND:Spinal muscular atrophy (SMA) is a frequent autosomal recessive disorder, characterized by lower motor neuron loss in the spinal cord. More than 95% of SMA patients show homozygous survival motor neuron 1 (SMN1) deletion. We previously developed a screening system for SMN1 deletion based on a modified competitive oligonucleotide priming-PCR (mCOP-PCR) technique. However, non-specific amplification products were observed with mCOP-PCR, which might lead to erroneous interpretation of the screening results. AIM: To establish an improved version of the mCOP-PCR screening system without non-specific amplification. METHODS: DNA samples were assayed using a new version of the mCOP-PCR screening system. DNA samples had already been genotyped by PCR-restriction fragment length polymorphism (PCR-RFLP), showing the presence or absence of SMN1 exon 7. The new mCOP-PCR method contained a targeted pre-amplification step of the region, including an SMN1-specific nucleotide, prior to the mCOP-PCR step. mCOP-PCR products were electrophoresed on agarose gels. RESULTS: No non-specific amplification products were detected in electrophoresis gels with the new mCOP-PCR screening system. CONCLUSION: An additional targeted pre-amplification step eliminated non-specific amplification from mCOP-PCR screening.
Authors: L M Brzustowicz; T Lehner; L H Castilla; G K Penchaszadeh; K C Wilhelmsen; R Daniels; K E Davies; M Leppert; F Ziter; D Wood Journal: Nature Date: 1990-04-05 Impact factor: 49.962
Authors: G van der Steege; P M Grootscholten; P van der Vlies; T G Draaijers; J Osinga; J M Cobben; H Scheffer; C H Buys Journal: Lancet Date: 1995-04-15 Impact factor: 79.321
Authors: Markus Feldkötter; Verena Schwarzer; Radu Wirth; Thomas F Wienker; Brunhilde Wirth Journal: Am J Hum Genet Date: 2001-12-21 Impact factor: 11.025