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Primed in situ labelling facilitates flow sorting of similar sized chromosomes
Author(s) -
Pich Uta,
Meister Armin,
Macas Jiří,
Doležel Jaroslav,
Lucretti Sergio,
Schubert Ingo
Publication year - 1995
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1046/j.1365-313x.1995.07061039.x
Subject(s) - biology , chromosome , propidium iodide , microbiology and biotechnology , ploidy , metaphase , karyotype , fluorescence in situ hybridization , genetics , gene , programmed cell death , apoptosis
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.