PRIMED IN-SITU LABELING FACILITATES FLOW SORTING OF SIMILAR SIZED CHROMOSOMES

Flow karyotyping and sorting of individual chromosome types is difficu lt when chromosomes of a complement do not differ sufficiently in DNA content. A strategy for sorting chromosomes of similar size has been d eveloped. For this purpose oligonucleotide primed in situ (PRINS)-labe lling was adapted to field bean chromosomes in suspension. With a prim er 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 (rep resenting DNA content) and fluorescein isothiocyanate signals (represe nting chromosome-specific label) allowed chromosomes deviating in leng th by less than 1% of the haploid metaphase complement to be sorted. T he average purity of sorted fractions was 95%. This technique should b e applicable also to chromosomes of other species for obtaining chromo some-specific painting probes, for construction of chromosome-specific libraries (both without additional DNA amplification), and for gene m apping.