Adapting in situ polymerase chain reaction for genotyping of cells in suspension

An approach is described for in situ polymerase chain reaction (ISPCR) base d on cycling primed in situ synthesis (PRINS) conditions defined for ct-sat ellite DNA. Using blood cell preparations subjected to limited fixation wit h paraformaldehyde, ISPCR cycling resulted in a gradual buildup of amplicon at the site of synthesis, as judged by the characteristic presence of pair ed nuclear spots corresponding to specific centromeres. Using longer cyclin g regimens, primers for single copy genes also generated paired nuclear spo ts in a primer-pair-specific manner. In this context, the amplification ref ractory mutation system (ARMS) was evaluated for in situ applications. In A RMS, allele-specific primers are used in such a manner that PCR proceeds on ly when an exact 3' match between annealed primer and template is recognize d by DNA polymerase. Using normal and mutant primers for the Delta F508 mut ation in the cystic fibrosis transmembrane conductance regulator (CFTR) gen e as a model system, it was not possible to reliably differentiate between ARMS reactions by accumulation of direct labeled reaction product in cells, because of ARMS-independent nonspecific labeling. However, by DNA extracti on and reamplification with ARMS primers, it was shown that amplicon accumu lates in cells in the expected primer/template-dependent manner crucial to mutation detection by ARMS. It was also shown that non-specific signal is d ue to primer dimer formation, especially in the absence of true template DN A. The impact of primer dimer formation in generating a false-positive sign al is discussed. The method described here enables a cell population to be analyzed for a given point mutation.