If you are just now reading this post I suggest skipping back to the preceding one to get a better understanding of “dark DNA”. This is a very busy picture, I know, but let’s take it one step at a time, ok? Explanatory text is below these pictures.
On either side of the microfuge tube at the top are two funny looking models of DNA replicons. If you’ve been reading my posts, you know what a replicon is. If not, you may want to dig back deeper into the blog.
Quick summation: A replicon is a piece of DNA where the initiation of DNA synthesis begins in a cell nucleus. There may be only one like in a bacterium or many of them in higher cells like our own. The number varies not only by species but also by the stage of the organism in embryonic development and perhaps beyond. The maximum number of replicons in cells like ours begins at conception and decreases as cellular differentiation begins to take place. If you peruse the blog you will see how replicon loss and the onset of differentiation may be occurring in the same sites on the DNA.
The model on the upper left is that of two DNA replicons joined together by an RNA bridge. The model on the upper right is that of two fused replicons where the bridge and any genetic material associated with it has been removed. The double replicon model represents an undifferentiated state whereas the fused model represents a differentiated state.
In the models below them, they are subjected to physical shearing (fragmentation) by vortexing samples in a mixture of aqueous (water-based) buffer together with a very caustic protein denaturant called phenol. The DNA may have been subjected to chemical breakage using chemical or enzymatic means prior to using the phenol. The ensuing double stranded fragments are shown just below these models.
Now pay very close attention here: Note that all the DNA not associated with the original RNA bridge in both models separates out in the aqueous phase. This is the stuff that most molecular biologists use to sequence DNA and put it back together like a giant jigsaw puzzle using overlapping sequences to figure out the correct alignment of sequences.
The bridge elements may have other kinds of molecules still associated with them like proteins and phosplipids known to be located near origins of replication on replicons. Even in the absence of such contaminants, these structures do not lend themselves to DNA sequencing because they are not simple linear DNA molecules. They have a higher order or tertiary structure.
So there therein lies the problem: Molecular biologists are trying to sequence DNA as if it were a simple thread. They break it up into small linear sequences which can randomly overlap with each other. They line them up to generate linear maps using these sequence overlaps. The problem with this approach is it only works if the DNA is actually a single continuous thread. Not only that, the molecular biologist simply throws out that which does not fit his model and cannot be sequenced. If replicons pair together like my models suggest, the differences could be staggering. It’s as if you were trying to explain a two dimensional painting using only one dimension, a straight line. Orders of magnitude of DNA superstructure could be overlooked as a result.
There will be another post very soon: Dark DNA, Part III.