Replication intermediates can be separated on agarose gels in two dimensions to reveal a wealth of data on mechanisms of DNA replication. When applied to mitochondrial DNA of higher vertebrates, this technique unearthed a host of unexpected findings, the full implications of which are still being absorbed. Here, we describe the procedures we use to isolate intact mitochondrial replication intermediates from liver of higher vertebrates and the process of separating DNA fragments on neutral two-dimensional agarose gels.
