REPEATED PRIMED IN-SITU LABELING - FORMATION AND LABELING OF SPECIFICDNA-SEQUENCES IN CHROMOSOMES AND NUCLEI

A new version of our previously published PRINS (PRimed IN Situ labeli ng) method is presented. It represents a significant improvement in th e detection of specific DNA sequences in situ. The idea is to perform the reaction repeatedly, rather than just once. This change of strateg y results in a localized accumulation of sequence-specific labeled DNA , resulting in up to a 15-fold amplification of the signal as compared to the standard PRINS method. Interestingly, the retention of the lab eled DNA is so good that it stays within the chromosomal band where it is synthesized, provided that the reaction is not performed an excess ive number of times. The key trick is the performance of the procedure on small glass slides in PCR tubes, thereby avoiding the use of cover slips. In addition, use of the small glass slides seems to give less variance when the signals are quantified in a fluorescence microscope.