n Applications in Genetics
Materials
Equipment LightCycler instrument
Reagents QIAmp blood kit (Qiagen, Hilden, Germany)
DNAzol (Life Technologies, Bethesda, MD, USA)
PCR primers (Life Technologies)
DNA Samples
PCR Primers and
Hybridization Probes
Optimizing PCR
Hybridization probes (TIB Molbiol, Berlin, Germany)
FastStart LightCycler kits (Roche Diagnostics)
Procedure
DNA, cell lines and paraffin-embedded tissue were anonymized samples from our
diagnostic laboratory. Lymphoblast cell lines were produced from peripheral
blood from patients and healthy blood donors, by EBV transformation. Tissue
samples from patients were from tumor and normal breast tissue.
DNA was isolated form peripheral blood using DNAzol (Life Technologies,
Bethesda, MD, USA) and from paraffin-embedded tissue using the QIAgen blood
kit (Qiagen) according to the manufacturer's instructions.
Primers and probes were designed using OLIG05 software (MBI, Cascade, CO,
USA). Some primers and probes are different from the ones we described before
[1,4]. The probes were designed to have melting temperatures (Tm) above the Tm
of the PCR primers and the Tm of the fluorescein-labeled probe (anchor probe)
above the Tm of the other probes. The probes were custom-made by TIB-MOLBIOL (Berlin, Germany). The sequences are given in Table 1.
PCR products for PCR optimization were prepared with DNA from peripheral
blood lymphocytes from healthy control subjects (wild type) and from patients
who were heterozygous for the mutations studied (2841G~T and 3159G~T in
BRCA1).
PCR reactions were performed according to the LightCycler kit instructions
(FastStart DNA Master SYBR Green I), using master mixes supplied in kits from
Roche Diagnostics with 5 picomoles of each primer per reaction in a 20-fJl reaction volume (25IlM). Using 10 ng of DNA per capillary, 35 PCR cycles were monitored with SYBR Green I. At the end of the PCR cycles, melting curve analysis
(LightCycler kit instructions) was performed by heating to 95°C for 2 min, followed by cooling to 65°C and gradual heating to 92°C at 0.2°C/s. The PCR reactions were optimized with respect to annealing temperature and [Mg2+ ], using
SYBR Green I, final Mg2+ concentrations from 2-5 mM and annealing temperatures around the T A suggested by the OLIG05 software.
For hybridization probe experiments, PCR reactions were set up according to
the LightCycler kit instructions (FastStart DNA Master Hybridization). Probes
were used at final concentrations of 0.1-0.4 11M. PCR conditions were derived
from the SYBR Green I experiments. Mg2+ was optimized again with the probes.
Materials
Equipment LightCycler instrument
Reagents QIAmp blood kit (Qiagen, Hilden, Germany)
DNAzol (Life Technologies, Bethesda, MD, USA)
PCR primers (Life Technologies)
DNA Samples
PCR Primers and
Hybridization Probes
Optimizing PCR
Hybridization probes (TIB Molbiol, Berlin, Germany)
FastStart LightCycler kits (Roche Diagnostics)
Procedure
DNA, cell lines and paraffin-embedded tissue were anonymized samples from our
diagnostic laboratory. Lymphoblast cell lines were produced from peripheral
blood from patients and healthy blood donors, by EBV transformation. Tissue
samples from patients were from tumor and normal breast tissue.
DNA was isolated form peripheral blood using DNAzol (Life Technologies,
Bethesda, MD, USA) and from paraffin-embedded tissue using the QIAgen blood
kit (Qiagen) according to the manufacturer's instructions.
Primers and probes were designed using OLIG05 software (MBI, Cascade, CO,
USA). Some primers and probes are different from the ones we described before
[1,4]. The probes were designed to have melting temperatures (Tm) above the Tm
of the PCR primers and the Tm of the fluorescein-labeled probe (anchor probe)
above the Tm of the other probes. The probes were custom-made by TIB-MOLBIOL (Berlin, Germany). The sequences are given in Table 1.
PCR products for PCR optimization were prepared with DNA from peripheral
blood lymphocytes from healthy control subjects (wild type) and from patients
who were heterozygous for the mutations studied (2841G~T and 3159G~T in
BRCA1).
PCR reactions were performed according to the LightCycler kit instructions
(FastStart DNA Master SYBR Green I), using master mixes supplied in kits from
Roche Diagnostics with 5 picomoles of each primer per reaction in a 20-fJl reaction volume (25IlM). Using 10 ng of DNA per capillary, 35 PCR cycles were monitored with SYBR Green I. At the end of the PCR cycles, melting curve analysis
(LightCycler kit instructions) was performed by heating to 95°C for 2 min, followed by cooling to 65°C and gradual heating to 92°C at 0.2°C/s. The PCR reactions were optimized with respect to annealing temperature and [Mg2+ ], using
SYBR Green I, final Mg2+ concentrations from 2-5 mM and annealing temperatures around the T A suggested by the OLIG05 software.
For hybridization probe experiments, PCR reactions were set up according to
the LightCycler kit instructions (FastStart DNA Master Hybridization). Probes
were used at final concentrations of 0.1-0.4 11M. PCR conditions were derived
from the SYBR Green I experiments. Mg2+ was optimized again with the probes.