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

For known mutations, real time polymerase chain reaction followed by meltin g curve analysis, using hybridization probes, is highly sensitive, rapid an d an efficient approach to mutation detection. We have used this approach o n the LightCycler(TM) for the detection of single base mutations in a singl e cell, without nested PCR. Hybridization probes were designed for two sequ ences in the BRCA1 gene containing 3 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 usi ng hybridization probes. The 5'-probes were 3'-labeled with FITC, whereas t he 3'-probes, covering the mutation, were 5'-labeled with LC-Red640 (wild t ype 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 reac tion mix was prepared in reaction capillaries and a single cell, picked by micromanipulation, was added to this: mix. The DNA from the cell is release d during the 5-min preheating step of the PCR, using the FastStart hybridiz ation kit (Roche). Reproducible results: were obtained, without the need of nested PCR. The technique is useful for microdissected tumors and, with ot her genes, has great potential for pre-implantation diagnosis in IVF and an alysis of residual disease in cancer. (C) 2001 Elsevier Science B.V. All ri ghts reserved.