Primed in situ labelling facilitates flow sorting of similar sized chromosomes

Flow karyotyping and sorting of individual chromosome types is difficult when chromosomes of a complement do not differ sufficiently in DNA content. A strategy for sorting chromosomes of similar size has been developed. For this purpose oligonucleotide primed in situ (PRINS)-labelling was adapted to field bean chromosomes in suspension. With a primer designed according to a tandemly repetitive sequence ( Fokl element) PRINS-labelling resulted in fluorescence signals specific in position and intensity for each chromosome. A bivariate sorting mode combining fluorescence pulse areas obtained from propidium iodide staining (representing DNA content) and fluorescein isothiocyanate signals (representing chromosome-specific label) allowed chromosomes deviating in length by less than 1% of the haploid metaphase complement to be sorted. The average purity of sorted fractions was 95%. This technique should be applicable also to chromosomes of other species for obtaining chromosome-specific painting probes, for construction of chromosome-specific libraries (both without additional DNA amplification), and for gene mapping.