Isolation of specific chromosomes and their DNA
Chromosome 21 is the only human chromosome in the mouse human hybrid, WA17. WA17 cells were treated with 0.1 μg/ml Colcemid, lysed, and mitotic chromosomes were separated based on their sizes in sucrose velocity gradients. The position of chromosome 21 on the gradient was determined in two ways. First, 1% of each fraction was denatured, blotted onto nitrocellulose, and quantitatively probed for a repetitive sequence found only in human DNA. Of the 76 fractions, fractions 44 through 62 contained human Alu sequences. Second, fractions containing human DNA were pooled in groups of three, Southern blotted, and probed with a chromosome 21-specific probe, 21.159. Pool E-DNA (fractions 54-56) hybridized most strongly to the 21.59 probe, while the control H DNA (fractions 34-36) and mouse L cell DNA failed to hybridize. Based on densitometric scans of autoradiograms, we estimate chromosome 21 DNA in pool E is 15-fold purified with respect to WA17 DNA. © 1985.
