(65b) Rapid, Free Solution Electrophoretic Separation of Long DNA

We have developed a rapid, free solution electrophoretic DNA separation technique for long DNA (greater than 1 kb) that is capable of separating up to 6 kb with 1 kb resolution, with the potential to theoretically separate up to 48 kb with hundred-base resolution in a matter of minutes.  In our system, nonionic micelles are transiently attached to the end of the DNA molecules and lower the DNA’s electrophoretic mobility in a length-dependent manner.  By tuning the size of the micelle, we can separate DNA in a specific size range.  Because the separation takes place in free solution rather than in a gel, the separation time is also extremely low.

Traditionally, sieving matrices in the form of gels have been used in capillary electrophoresis to perform length-based separation of DNA molecules.  However, long DNA molecules cannot be separated in these traditional gels because the DNA undergoes biased reptation, aligning in the direction of the electric field and migrating with a length-independent mobility.  In the 1980s, pulsed-field gel electrophoresis was developed which increased the length of DNA that could be separated to the millions of base pairs, but has the disadvantage of requiring several days for separation.  Techniques that have been developed in the past decade to separate long DNA include top-down approaches such as nanofabricated pillars, and bottom-up approaches such as self-assembled magnetic particles.  The major drawbacks to these methods include the time consuming and expensive nature of nanofabrication processes, and the low resolution capabilities of the matrices.  Instead of single base resolution achieved with traditional gels, these artificial matrices can typically only resolve DNA molecules that differ in length by thousands of bases. There are many applications such as genome mapping and DNA fingerprinting which involve separating DNA thousands of bases long, and improving the resolution of these separations is critical.