Mammalian Metaphase Chromosomes with High Molecular Weight DNA isolated at pH 10.5

Abstract

MAMMALIAN metaphase chromosomes may be isolated either at an acid pH^1–4 or at nearly neutral pH^5–7. The only exception is the method of Corry and Cole⁸, performed at pH 9.5. We used alkaline sucrose velocity gradients to determine the size distributions of DNA (molecular weight) in metaphase chromosomes in an attempt to understand their arrangement. Initial experiments yielded 1 × 10⁶ molecular weight DNA (single stranded) pieces regardless of chromosome size. When metaphase cells are lysed directly on the gradient a high molecular weight (1 × 10⁸ for single stranded) is obtained. We therefore examined a large number of chromosome isolation procedures and cytological conditions to determine their effects on the molecular weight of the DNA.