Detection of a Single Base Substitution
in Single Cells by Melting Peak Analysis
Using Dual-Color Hybridization Probes
GERARD PALS*
Introduction
Detection of point mutations in very small amounts of DNA in our lab is performed in micro dissected material from tumors and small premalignant lesions
to assess loss of mutant or wild-type alleles of tumor suppressor genes. Even more
demanding is the detection of point mutations in a single cell, which is required
in preimplantation genetic diagnosis.
Real-time PCR followed by melting curve analysis, using hybridization
probes, is highly sensitive, rapid and an efficient approach to mutation detection. We have used this approach on the LightCyder instrument for the detection of single-base mutations in a single cell, without nested PCR [1]. The presence of mutant and wild-type alleles was assessed using a single FITC-labeled
anchor probe and wild-type and mutant-specific detection probes with different labels (LCRed640 and LCRed705).
Hybridization probes were designed on sequences in the BRCAl gene, covering mutations that are present in cell lines available in our lab. PCR reactions of
small fragments (100-300 bp) containing the probe sequences were optimized
using the SYBR Green I, before using hybridization probes. The 5' -probes were
3' -labeled with FlTC, whereas the 3' -probes, covering the mutation, were 5'labeled with LCRed640 (wild-type probes) or LCRed705 (mutant probes). Dualcolor detection of wild-type and mutant sequence in a single tube was tested on
single cells. The reaction mix was prepared in reaction capillaries and a single
lymphoblast cell, picked by manual micromanipulation, was added to this mix.
The DNA from the cell is released during the 5-min preheating step of the PCR,
using the FastStart hybridization kit (Roche Diagnostics, Mannheim, Germany). Reproducible results were obtained, without the need of nested PCR.
The technique is useful for micro dissected tumors and, with other genes, has
great potential for preimplantation diagnosis in IVF and analysis of residual
disease in cancer .
.. Gerard Pals (~) (e-mail: g.pals@vumc.nl).Department of Clinical Genetics,
Vrije Universiteit Medical Center,Amsterdam, The Netherlands, Van der Boechorststraat 7,
1081 BT Amsterdam, The Netherlands
•
in Single Cells by Melting Peak Analysis
Using Dual-Color Hybridization Probes
GERARD PALS*
Introduction
Detection of point mutations in very small amounts of DNA in our lab is performed in micro dissected material from tumors and small premalignant lesions
to assess loss of mutant or wild-type alleles of tumor suppressor genes. Even more
demanding is the detection of point mutations in a single cell, which is required
in preimplantation genetic diagnosis.
Real-time PCR followed by melting curve analysis, using hybridization
probes, is highly sensitive, rapid and an efficient approach to mutation detection. We have used this approach on the LightCyder instrument for the detection of single-base mutations in a single cell, without nested PCR [1]. The presence of mutant and wild-type alleles was assessed using a single FITC-labeled
anchor probe and wild-type and mutant-specific detection probes with different labels (LCRed640 and LCRed705).
Hybridization probes were designed on sequences in the BRCAl gene, covering mutations that are present in cell lines available in our lab. PCR reactions of
small fragments (100-300 bp) containing the probe sequences were optimized
using the SYBR Green I, before using hybridization probes. The 5' -probes were
3' -labeled with FlTC, whereas the 3' -probes, covering the mutation, were 5'labeled with LCRed640 (wild-type probes) or LCRed705 (mutant probes). Dualcolor detection of wild-type and mutant sequence in a single tube was tested on
single cells. The reaction mix was prepared in reaction capillaries and a single
lymphoblast cell, picked by manual micromanipulation, was added to this mix.
The DNA from the cell is released during the 5-min preheating step of the PCR,
using the FastStart hybridization kit (Roche Diagnostics, Mannheim, Germany). Reproducible results were obtained, without the need of nested PCR.
The technique is useful for micro dissected tumors and, with other genes, has
great potential for preimplantation diagnosis in IVF and analysis of residual
disease in cancer .
.. Gerard Pals (~) (e-mail: g.pals@vumc.nl).Department of Clinical Genetics,
Vrije Universiteit Medical Center,Amsterdam, The Netherlands, Van der Boechorststraat 7,
1081 BT Amsterdam, The Netherlands
•