Introducing Shotgun DNA Mapping

Before I got immersed in the world of deuterium-less water, I was a rabid DNA-man. The project was Shotgun DNA Mapping which was a term we invented to describe a quick protocol for mapping a DNA sequence.

In theory, the technique was awesome: unzip a DNA sequence with optical tweezers and compare the data to a library of simulated data for a given genome to figure out where you are in the genome. This would lead to a bigger project called Shotgun Chromatin Mapping which was the similar except you could map protein locations on DNA fragments using the same technique.

For this to work, you need three components:

  1. Optical Tweezers – to unzip DNA and record data
  2. DNA – to unzip
  3. A computer simulation – to simulate DNA unzipping and match recorded data to simulated data

I dedicated a few blog posts in my other blog to discussing the basic principles of the project in case you need to get caught up to speed. But in case you don’t have time for all that, here is the whirlwind summary:

Optical tweezers are an optical system that requires a laser, a microscope objective, a condensor, some steering components, and a detector (in our case a quadrant photo diode) among other things. The laser is focused by the objective and this focus can exert forces on tiny dielectric particles. Our particles are microspheres. (The blog posts linked explain the physics of this in great detail.)

Using some principles of biochemistry I can attach a microsphere to a DNA fragment that is specially designed to: (1) tether to a glass slide using antibody-antigen interactions, (2) contain a weak point in the DNA backbone to begin unzipping, and (3) be versatile enough to use a variety of different DNA sequences.

I can then tether the DNA to slides and place them in the path of the laser. The focus will attract the beads, and if the tethering process works properly then the beads will be attached to DNA. This is how we are able to exert forces on the DNA. Our detector is used to track the laser movement, and those signals get converted into force data. The forces recorded are on the order of pN, which is insanely small but enough to distinguish from background noise.

Once we have unzipping data we can use a computer program to compare this information to a library of simulated unzipping data for a genome. In our proof of principle study we used the yeast genome, so we simulated unzipping fragments for the entire genome and then used actual yeast genomic DNA to unzip.

Unfortunately I hit an impassable road block in the experiment. The DNA I created wouldn’t unzip. I tried everything I could think of, reworked the entire process and tried to come up with alternate methods for creating the DNA fragments. Ultimately I had to switch to the project I’m working on now…

…But that doesn’t mean that the project was a complete failure. I’m sure the protocols and techniques I employed can be useful to someone, somewhere, someday and so I’m going to highlight posts from my old notebook here as a way to kind of direct attention to the protocols that summarize my project well and were most useful for me.

In this way, one wouldn’t need to sift through mounds of information just to find one thing. And it would provide visitors here a little more information about my background and something I keep alluding to. All in the name of open science!