Making DNA lay down straight
The whole linchpin of this process is being able to measure the length of the strands of DNA (more accurately, the lengths of DNA fragments but we'll get to that shortly). In order for length to have any meaning, we need to have the subjects we're measuring be as close to a straight line as possible. In order to do that we use a glass surface (you remember those microscope slides you used in high school) and a cover slip that has microscopically small channels carved into it. The DNA is placed, in solution, onto this surface and, using a magical process I know nothing about, the DNA is stretched out along those channels which serve as guides for straightening out the molecules.
Cutting it up into fragments
In order to create meaningful maps that can be used to identify and compare organisms we need to break up the DNA molecules into fragments. It's these fragments that we measure to create that nice-looking barcode map. In order to create those maps you use what is called a "restriction enzyme", which are enzymes that actually cut DNA. A particular restriction enzyme always cuts in the same place, at a particular occurrence of base pairs in the DNA strand. For example, the enzyme BamHI cuts at restriction sites of GGATCC. As an aside, most restriction enzymes cut at sites that are palindromic.. there's no obvious reason that I know of why that is, but it sure is a neat coincidence. Due to the genetic makeup of different organisms, different enzymes will cut different numbers of fragments for each organism. Part of our process is picking an enzyme that will cut the "right number" of fragments, that is, enough to make a meaningful map. Too few fragments or too many fragments often make the maps indistinguishable from each other.
Measuring those fragments
As part of the preparation of the DNA, a stain is applied that will cause the fragments to light up or "fluoresce" when exposed to a laser. The glass slip containing the DNA solution is placed on a fluorescent microscope that has a camera attached to it and an automated software system moves the camera up and down the length of those channels and takes pictures through the microscope. Here's an example of what it looks like. Remember this is one tiny fraction of a single image from the microscope. You can see several broken strands of DNA. The colored one is one that has been picked out by the software as clean enough to be measured and recorded.
Hundreds of such images are acquired and finally fed through some image processing software that finds the nicest looking DNA molecules and it finds the fragments and measures their length in some unit of measure that is smaller than anything I can image. Finally those fragment lengths are recorded in order and they can be visually represented by that barcode-like display I showed last time. Here's an artists interpretation of how that looks:
Assembling the pieces
Here's where the real world comes in and whacks you in the head. DNA will almost never stay fully in tact throughout the process I just described. And even if it did, there are usually a lot of molecules and they tend to overlap each other or they don't straighten out exactly right (or sometimes at all). So what you end up with is lots and lots of small maps that represent just a chunk of the entire strand of DNA. And here is where some intense computing power is brought to bear on the problem as we take all of those smaller maps and try and determine how and where the overlap each other. It's kind of analogous to trying to put a piece of paper back together after it has been through a shredder. When this process is finally done (and everything worked out okay) you end up with a "consensus map", which is the amalgamation of all of those smaller map chunks.
Once you've got that consensus map, then the doors open to a wide range of things you can do with it and that's where the software that I'm working on comes into play. But I'll save that stuff for the third and final installment of this series. Thanks for reading!
