DNA separations
Bill Bernhard’s group approached us about using pnc-Si membranes to separate DNA species by diffusion. Bill’s lab studies the effects of ionizing radiation (ex. X-rays, gamma rays, UV light) on DNA using electron paramagnetic resonance (EPR). There are three major species of DNA that they wish to study using our membranes: short oligonucleotides, larger supercoiled plasmids, and spacially larger nicked plasmids.
By my naive understanding, as DNA is damaged, the plasmids may first become nicked and then small oligonucleotides will break off. Bill’s group wants to damage the DNA and then filter it using our membranes to find what size pieces will break off.
To observe the DNA they are using UV absorbance, since DNA bases give off a strong signal. This method will tell you the presence and concentration of DNA, but not the size. Presumably they can use agarose gel electrophoresis to find the sizes as I use SDS-PAGE to study proteins. The first controls I helped them to set up were fairly simple. I applied their sample of only supercoiled DNA to a membrane (w620) and they showed by UV absorbance that it did not pass through. They estimated that the size of the supercoiled DNA was 20nm. It appears that the largest pores in w620 are about 35nm. I then applied a sample of only oligonucleotide to a membrane from w620, and they showed that this did pass through.
So we know that w620 should be able to potentially separate these two species. We have not performed that experiment yet, and it may be hard to quantify by a gel because the supercoiled DNA is so much larger than the oligo and both may not be resolved well in the same gel. They do however have a DNA ladder and are interested in separating this (as am I!).