Primed in situ labelling facilitates flow sorting of similar sized chromosomes

Publisher Website
Google Scholar
Abstract
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.

This publication has 0 references indexed in Scilit: