m Applications in Genetics
Materials
Equipment LightCycler instrument (Roche Molecular Diagnostics, Japan)
Reagents M-MLV reverse-transcriptase (Gibco BRL)
Random primers (TAKARA, Japan)
RNase-inhibitor (Wako, Japan)
pGEM-T easy plasmid (Promega)
Amplification primers (Nihon Gene Research Laboratories Inc., Sendai, Japan)
Hybridization probes (Nihon Gene Research Laboratories Inc.)
High Pure PCR Template Purification Kit (Roche Molecular Diagnostics)
LightCycler-DNA Master SYBR Green I (Roche Molecular Diagnostics)
LightCycler-DNA Master Hybridization Probes (Roche Molecular Diagnostics)
Procedures
For the detection of a 2-bp deletion using the hybridization probe-based method,
we followed the procedure described in the next section.
Sample Preparation The patient was a I5-year-old boy with classic Fabry disease who suffered from
angiokeratoma, acroparesthesias, and attacks of pain in his legs. We had already
identified a 2-bp deletion mutation at nucleotide 11008-11009 in his GLA gene
(GenBank Accession #XI4448) by RT -PCR and sequencing [4, 5]. This change led
to a frameshift, which was described previously in another patient with the same
disease [6]. We extracted genomic DNA from peripheral blood lymphocytes of
the patient, his mother, his unaffected brother, and his maternal grandmother,
using the High Pure PCR Template Purification Kit according to the manufacturer's instructions.
Oligonucleotides For fluorescence PCR analysis, we prepared two PCR primers and two fluorescence probes (Table O. A 25-mer oligonucleotide probe, Anchor Probe, was
labeled at the 5' end with the LCRed640 fluorophore, which was modified at the
3' end by phosphorylation to avoid extension. The other 20-mer oligonucleotide probe, Mutation Detection Probe, was synthesized to anneal the 2-bp
deletion mutation region, which was labeled at the 3' end with fluorescein. The
distance between the two probes was one oligonucleotide. When both probes
hybridize in close proximity, fluorescence resonance energy transfer occurs,
producing a specific fluorescence emission of LCRed640 as a result of fluorescein excitation.
Materials
Equipment LightCycler instrument (Roche Molecular Diagnostics, Japan)
Reagents M-MLV reverse-transcriptase (Gibco BRL)
Random primers (TAKARA, Japan)
RNase-inhibitor (Wako, Japan)
pGEM-T easy plasmid (Promega)
Amplification primers (Nihon Gene Research Laboratories Inc., Sendai, Japan)
Hybridization probes (Nihon Gene Research Laboratories Inc.)
High Pure PCR Template Purification Kit (Roche Molecular Diagnostics)
LightCycler-DNA Master SYBR Green I (Roche Molecular Diagnostics)
LightCycler-DNA Master Hybridization Probes (Roche Molecular Diagnostics)
Procedures
For the detection of a 2-bp deletion using the hybridization probe-based method,
we followed the procedure described in the next section.
Sample Preparation The patient was a I5-year-old boy with classic Fabry disease who suffered from
angiokeratoma, acroparesthesias, and attacks of pain in his legs. We had already
identified a 2-bp deletion mutation at nucleotide 11008-11009 in his GLA gene
(GenBank Accession #XI4448) by RT -PCR and sequencing [4, 5]. This change led
to a frameshift, which was described previously in another patient with the same
disease [6]. We extracted genomic DNA from peripheral blood lymphocytes of
the patient, his mother, his unaffected brother, and his maternal grandmother,
using the High Pure PCR Template Purification Kit according to the manufacturer's instructions.
Oligonucleotides For fluorescence PCR analysis, we prepared two PCR primers and two fluorescence probes (Table O. A 25-mer oligonucleotide probe, Anchor Probe, was
labeled at the 5' end with the LCRed640 fluorophore, which was modified at the
3' end by phosphorylation to avoid extension. The other 20-mer oligonucleotide probe, Mutation Detection Probe, was synthesized to anneal the 2-bp
deletion mutation region, which was labeled at the 3' end with fluorescein. The
distance between the two probes was one oligonucleotide. When both probes
hybridize in close proximity, fluorescence resonance energy transfer occurs,
producing a specific fluorescence emission of LCRed640 as a result of fluorescein excitation.