Chromosome-Specific PRINS

The identification of individual chromosomes is of great importance in cytogenetics, in order to detect aneuploidies or chromosomal rearrangements associated with genetic diseases. This can be achieved by several techniques based either on the intrinsic staining properties of the chromosomes in producing bands (the banding pattern being specific for each pair of chromosomes) (1 ) or the use of a DNA probe to detect specifically a region of the chromosome by fluorescence in situ hybridization (FISH) (2 ). The use of centromeric α satellite sequences as FISH probes is very popular because of the specificity of these sequences. α Satellite (or alphoid) DNA is a family of tandemly repeated sequences present at the centromere of all human chromosomes (3 ). Subfamilies, some of them specific for one or a small group of chromosomes, can be identified within alphoid DNA both by the periodic distribution of restriction sites and the nucleotide sequence of the 171-bp basic motif (4 ). These chromosome-specific subfamilies can therefore be used as FISH probes. This approach is limited, however, since the DNA sequences of some subfamilies are very close to each other, and crosshybridization can occur between the centromeric sequences of several pairs of chromosomes. This is the case with chromosomes 13 and 21, for example, which share 99.7% homology in their alphoid sequences (5 ). The development of the primed in situ (PRINS) technique of labeling DNA (6 –8 ) introduced a solution to this problem. The PRINS procedure consists of the use of a small oligonucleotide (usually 18–22 nucleotides) from the sequence of interest as a primer. The primer is annealed to the denatured DNA of a chromosome or cell preparation.