Detection of a single base substitution in a single cell using the LightCycler™

Abstract

For known mutations, real time polymerase chain reaction 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 LightCycler™ for the detection of single base mutations in a single cell, without nested PCR. Hybridization probes were designed for two sequences in the BRCA1 gene containing a single base substitution and deletion, respectively. Polymerase chain reactions of small fragments (100–200 bp) containing the probe sequences were optimized using SYBR Green1, before using hybridization probes. The 5′-probes were 3′-labeled with FITC, whereas the 3′-probes, covering the mutation, were 5′-labeled with LC-Red640 (wild type probes) or LC-Red705 (mutant probes). Dual color detection of wild type and mutant sequences in a single tube was tested on single cells. The reaction mix was prepared in reaction capillaries and a single cell, picked by 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). Reproducible results were obtained, without the need of nested PCR. The technique is useful for microdissected tumors and, with other genes, has great potential for pre-implantation diagnosis in IVF and analysis of residual disease in cancer.