Bright-Field Microscopic Detection of Oligonucleotide PRINS-Labeled DNA in Chromosome Preparations

Primed in situ (PRINS) labeling has become an alternative to in situ hybridization (ISH) for the localization of nucleic acid sequences in cell (1 –4 ) and tissue preparations (5 ; see also . Chapter 5 )In the PRINS method, an unlabeled primer (restriction fragment, PCR product, or oligonucleotide) is annealed to its complementary target sequence in situ . The primer serves as an initiation site for in situ chain elongation using a thermostable DNA polymerase and labeled nucleotides, which can be detected directly by fluorescence microscopy, such as fluorochrome-labeled dNTPs, or indirectly using, e.g., biotin- or digoxigenin-dUTP and the application of fluorochrome-conjugated avidin or antibody molecules (3 ,6 ,7 ). The detection limit of the PRINS technique appears to be on the order of low-copy sequences (3 ,8 .