Chromatid contamination can impair the purity of flow-sorted metaphase chromosomes.
We describe a phenomenon where a second flow karyotype is superimposed on the normal metaphase flow karyotype of human lymphoblastoid chromosomes. The events of this second flow karyotype contain half the DNA content of corresponding events in the normal flow karyotype and, when sorted, show the morphology of single chromatids. DNA amplified by degenerate oligonucleotide-primed PCR of 300 chromatids sorted from a single peak and hybridized onto normal metaphase spreads painted the entire length of the corresponding chromosome type. In retrospect, we could observe this phenomenon at a low level in 37% of randomly selected, previously analyzed flow karyotypes and in several published flow karyotypes from other laboratories. Experimentally, chromatid frequency was shown to vary with the colcemid blocking conditions as well as with the cell line used. At the extreme, we obtained a 4:10 ratio of chromatids to metaphase chromosomes, whereas this could be reduced tenfold by altering the colcemid blocking conditions in the same cell line. The presence of chromatids in chromosome preparations has important implications for the purity of isolated fractions used for generating libraries and in PCR analysis.
Telenius
H
H
Department of Pathology, University of Cambridge, United Kingdom.
de Vos
D
D
Blennow
E
E
Willat
L R
LR
Ponder
B A
BA
Carter
N P
NP
D002460:Cell Line; D002842:Chromatids; D002875:Chromosomes; D005434:Flow Cytometry; D006801:Humans; D007621:Karyotyping / Q000379:methods*; D008677:Metaphase; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction