Re: How can a contour length of an isolated covalently closed circular (ccc) DNA molecule be measured?

Hi,

Thanks for your question. Measuring the contour length of DNA isn't an exact art. I know only too well from years of doing so using transmission electron microscopy and atomic force microscopy. In general, you must be aware that the length of the DNA depends upon the physical state of the DNA, its hydration state, the presence of intercalators (such as ethidium bromide) and the surface to which it adsorbed.

You can measure the contour length if you are lucky enough to be viewing the microscope image through some software, as is typical with AFM, but I will assume that you are not. Do you, in this case, have access to facilities to photograph your microscope image, in which case any measurement of the micrograph can be multiplied by the magnification at which the image was taken.

It is important to note that ccc DNA can either be supercoiled or relaxed. Being ccc simply means that it is covalently closed, and that the duplex is continuous, but it can either be supercoiled or relaxed. If supercoiled, it won't be possible to meaure the contour length of the DNA.

Various enzymatic reactions can result in a state when you have covalently-closed DNA, but without supercoiling, i.e relaxed. In this state it would in theory be possible to measure contour length, but this would also be pretty much the same as open-circular form DNA, i.e if just one stand of the DNA is nicked.

However, probably the best way is to linearise the DNA, if this is possible in your experiment?

Also bear in mind that when viewing molecules through a microscope you are seeing a 2D image, thus looping out of the DNA away from the surface, towards your eyes, will make the DNA seem shorter than it is.

In terms of standards, simply using an OC or linear plasmid of known size, and taking an average length of these molecules, should be sufficient.

Hope this helps,

Jim
MAD Scientist