Flow Cytometry flow sorting cells for PCR
DRAQ5 directly and stoichiometrically reports DNA content in cells, live or fixed.
Stained cells can be gated and sorted into isolated populations with different DNA content relating to the cell cycle, such as elevated G2/M or aneuploidy.
The DNA from the cell fractions can then be used directly for population-restricted PCR amplification.
Because DRAQ5 is a far-red emitting DNA dye this can be easily multiplexed with other parameters, like cell surface phenotype.
In a remarkable paper, Douwes Dekker, et al. (2004) extracted cells from paraffin-embedded sections, stained them with a fluorescent antibody and with DRAQ5. Cells were sorted into antibody positive and aneuploid or G2/M positive fractions. The DNA was extracted and subjected to single exon PCR and restriction digest to detect the presence of a mutation in each of the sorted fractions - SIMPLY BRILLIANT!
REFERENCE:
Multiparameter DNA flow-sorting demonstrates diploidy and SDHD wild-type gene retention in the sustentacular cell compartment of head and neck paragangliomas: chief cells are the only neoplastic component.
Pieter B Douwes Dekker, Willem E Corver, Pancras CW Hogendoorn, Andel GL van der Mey and Cees J Cornelisse.
J Pathol 2004; 202: 456–462 (view abstract)
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