Polymer Physics at uOttawa       ||       Physique des Polymeres uOttawa

Mechanisms of DNA Blocking in a Gel

David Sean

Gel electrophoresis of DNA has proven itself as a reliable technique for separating DNA fragments by size. This works by forcing DNA fragments to travel though a gel by the means of an externally applied electrical field (DNA is negatively charged, so it feels a force). The smaller DNA fragments can move though the gel easier than the bigger ones --- thus we have size-separation.

However, sequence-dependent separation is DNA is also possible via gel electrophoresis. Let me explain. Under certain conditions, the double-stranded structure of DNA breaks down and the strands begin to separate. This can be initiated by increasing the temperature beyond a certain threshold. It turns out that this denaturation --- or "melting" --- of the double stranded structure causes a migrating DNA fragment to drastically slow down in the gel. Thus fragments can be separarted based on the degree by which is is denatured.

The pairing of the DNA bases is responsible for the double helix, and since A-T basepairs are weaker than G-C basepairs, the degree by which a fragment is denatured will depend on its sequence! Sequence separation on DNA in this manner enables scientists to quickly identify different strains of virii or bacteria. The evolution (genetic mutations) of bacteria can be observed as they occur.

Unfortuneately, little is known about how/why the DNA fragments stop migrating in the gel when it starts to denature. Thus is it very diffucult to tweak the gel/experimental conditions in order to control how the fragments will block. To complicate matters DNA can partialy denature in two very different ways: 1) a denatured zone can form at the ends of the fragments, the picture above shows two split ends; or 2) This can be somewhere in the middle of the fragment, these are called "bubbles"). Do these different ways of denaturing affect blocking in the gel in the same manner? Which is more efficient at blocking?

Related Links:


2011      Gel electrophoresis of partially denatured DNA: split-ends, bubbles, and squids
2011      Physical interpretation of the Lr parameter in the theory for the gel electrophoresis of partially denatured DNA
2009      DNA Electrophoresis: how partially denatured DNA stops moving in a gel
2008      Quantitative predictions for DNA two-dimensional display according to size and nucleotide sequence composition


Contact       ||       Gary Slater : gslater@uottawa.ca       ||