Motion of DNA during gel electrophoresis

1. Constant field Electrophoresis Links to an external site. is one the most commonly used techniques for sequencing DNA molecules. We will explore how it works by means of a simulation. The simulation Links to an external site. investigated here is similar to an experiment of Volkmuth and Austin Links to an external site. but also has physics closely related to that of electrophoresis in agarose gel.

In the file hw6/elect.py, there are two main parameters to explore. The externally applied force, f_ext which we will take to be proportional to the electric field. The second is the length of the DNA.

1 Described the reason why it is useful in biology to perform electrophoresis of DNA.
- In what circumstances is it better to use agarose as a gel and where is polyacrylamide gel used?
- Is there are difference in behavior or linear DNA as opposed to circular DNA. Will the topology of circular DNA influence its mobility?

2 For n = 32, run the simulation and observe the motion. How does this compare to experimental observations? (Note that even when green pillars are not displayed, the periodic array still persists.) Note that the motion isn't like that of a snake and seems to be more episodic. Describe the different stages of motion qualitatively.

3 Calculate the mobility, that is the average velocity divided by field, for a variety of electric fields and chain lengths. For constant field, what happens as you vary chain length? There are many ways to do that. The easiest is just to run the simulation for a long time and measure the total displacement.

4 What does this imply about the use of this technique when the chain length becomes long for characterizing DNA?

To speed up the simulation you can change the display_interval parameter, or set the graphics flag to False.

2. Pulsed Field Electrophoresis Now we will study what happens when the field applied varies in time, which is called "Pulsed Field Electrophoresis". There are many variants of this technique, and they allow the differentiation of much longer chains than when an applied field is constant. One of the simplest conceptually to understand is Field Inversion Pulsed Electrophoresis. The field might go forwards for 2 seconds and backwards for one second and this cycle repeats itself. The file hw6/pulsed.py modifies the the electrophoresis simulation to allow pulsed fields. An extra function was added pulsed_f_ext that allows you to set the duration and strength of the pulsed fields. You can set this to simulate a variety of different geometries, but in this problem, we'll stick to only trying field inversion where the field is always parallel or anti-parallel to a single direction.

Experimentally, as a function of pulse time, there is a dip in the mobility that depends on chain length. Conversely, keeping pulse time constant and varying length leads to a dip in the mobility as a function of length and this can be used to separate long DNA chains

Run pulsed.py without changing any parameters. Run it for a long time, long enough to get a feel for how the pulsed field is modifying the dynamics. Now decrease the pulse time. Can you use this to understand why there is a dip in mobility as a function of pulse time? You've got to run this for long enough, and look at what is happening for pulse times where it slows down a lot. It's not that subtle if you play with the simulation for long enough, but it was a big mystery before this system was simulated. A student without much physics background can see what's happening as well.

Why is pulsed field gel electrophoresis useful, and in what situations is it used? State what advantage it has over the constant field case.
In order to do genetic sequencing, many techniques rely on the DNA being cut at reproducible points. How is this achieved in practice?