Flow analysis and sorting of microchromosomes (<3 Mb).
BACKGROUND
The analysis and isolation of high numbers of chromosomes smaller than 3 Mb in size (microchromosomes) with good purity is dependent primarily on the detection sensitivity of the flow cytometer and the precision of the sort unit. The aim of this study was to investigate the capability of using a conventional flow cytometer for the detection and sorting at high purity microchromosomes with an estimated size of 2.7 Mb.


METHODS
Chromosomes were isolated from a human cell line containing a pair of X-derived microchromosomes, using a modified polyamine isolation buffer. The chromosome preparation was labeled with Hoechst and Chromomycin and analyzed and purified using a MoFlo sorter (DAKO) configured for high-speed sorting. The purity of the flow-sorted microchromosomes was assessed by reverse chromosome painting.


RESULTS
Improved resolution of the peak of microchromosomes in a bivariate plot of Hoechst versus Chromomycin fluorescence was obtainable after discriminating clumps and debris based on gating data within a FSC versus pulse width plot.


CONCLUSIONS
Chromosomes of smaller size, less than 3 Mb, can be detected with high resolution and flow-sorted with high purity using a conventional flow sorter.
Ng
Bee L
BL
The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, United Kingdom. bln@sanger.ac.uk
Yang
Fengtang
F
Carter
Nigel P
NP
D001562:Benzimidazoles; D002021:Buffers; D045744:Cell Line, Tumor; D014128:Chromomycin A3; D020223:Chromosome Painting; D041321:Chromosomes, Human, X; D004247:DNA / Q000302:isolation & purification*; D005354:Fibrosarcoma / Q000737:chemistry* / Q000235:genetics / Q000473:pathology; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007621:Karyotyping / Q000379:methods*; D011073:Polyamines / Q000737:chemistry; D015203:Reproducibility of Results
D001562:Benzimidazoles; D002021:Buffers; D005456:Fluorescent Dyes; D011073:Polyamines; C447680:hoechst 32258; D004247:DNA; D014128:Chromomycin A3