Direct hybridization of sorted human chromosomes: localization of the Y chromosome on the flow karyotype.

Abstract

A method is described for directly hybridizing a small number of sorted chromosomes with specific DNA probes. The chromosomes are analyzed by flow cytometry and sorted by deflecting the droplets containing the desired chromosomes onto a nitrocellulose filter. By using probes specific for the human Y chromosome, it has been possible to unambiguously identify the peak corresponding to the Y chromosome in the flow karyotypes of a variety of male cell lines. The position of this peak was found to vary significantly from individual to individual, correlating with the heterochromatin chromosomal polymorphism of the human Y chromosome. The sensitivity of the hybridization was such that, with a probe for a male-specific repetitive sequence, only 2,500 sorted chromosomes were enough to obtain a clear, positive signal; 10,000 were needed with .a probe specific for a weakly repeated (maximum, 3-fold) sequence of Y chromosome. With this new method, chromosome sorting may be a rapid and efficient way to assign DNA sequences to chromosomes.

Chromosome sorting of primates: Isolation and identification of Y chromosome in green monkeys

“…We were not able to detect specific band by silver staining of the gel when template DNA was prepared from 5,000 chromosomes (lane 1). The similar detection sensitivity for a single copy gene has been reported by using radioactive probes for Southern blotting of sorted-out chromosomes (BERNHEIM et al, 1983). On the other hand, S R Y primers did not yield PCR product with the template D N A derived from peak " a " and " b " even large number of chromosomes (105 ) was collected (lane 3).…”

High-Resolution Chromosome Sorting and DNA Spot-Blot Analysis Assign McArdle's Syndrome to Chromosome 11

“…Finally, filter panels that are co'nstructed earlier may be tested and retested rapidly to map up to five DNA probes. Chromosome sorting has been used to identify a Y-specific sequence (19). However, by using a dual laser sorter' and improved methods, we are now able to map a gene to any one of 21 human chromosomes in a single hybridization step.…”

Construction and characterization of a DNA library from mouse chromosomes 19 purified by flow cytometry

“…The second group contains 1 clone ( [6], fig 3B) which hybridizes with almost all of the chromosomes, but with a preferential recognition of areas d to j as compared to the area a corresponding to chromosome 19. This pattern is very different from the reference one : therefore we cannot identify clone 6 as originating from chromosome 19.…”