Confusing Computers

2/27/12
Today Dr. Kirchman did not work in the lab, but on his computer. The DNA we had sent off was back and sequenced so we spent the day cleaning up the data. To sequence DNA, a fluorescent dye is given to each nucleotide. A computer measures the wavelength each nucleotide produces, and using these wavelengths, can determine the nucleotide that goes with the corresponding color. Primers run in opposite directions that read the DNA sequence. Two primers are used because it takes a little while for a primer to get going so the wavelengths of the nucleotides are really unclear in the beginning. Therefore, if you have two primers that are unclear in the beginning, but are clear at the end and they run in opposite directions, you will hopefully be able to get a clear idea of what the nucleotides are.  However, since our DNA samples of the rail birds were so small, the quality of our DNA wasn't too great, leading to the wavelengths of the nucleotides looking a bit like this:

when in a perfect world, they should look like this:

Basically, what I did today was edit the base pairs. When the computer cannot recognize whether the wavelength is a C or a G, there are other letters (for example R, K, and W) that represent a nucleotide that can be a C or a G or an A or a G. I went through the DNA and tried to make my best guess about what nucleotide matched the wavelength based on the prettier looking primer. Sometimes both wavelengths were messy and had double peaks really close to one another in which case, I would use one of the letters that represented that the exact nucleotide was unclear.
After going through all the base pairs, I then had to cut off the primers at the ends of the section of DNA. I did this by taking the sequenced primers and having the computer match them up at the ends of the sequence. I then simply removed the bases that overlapped with the primer. Lucky for me, the four DNA sequences I did were relatively clean and the section of DNA that Dr. Kirchman and I are using to examine the rail birds is relatively small. I am not going through the entire rail genome (which would take forever to do), but simply a small section of the DNA. Thankfully, I also did an okay job of my DNA cleaning up skills because when Dr. Kirchman and I quickly looked at the sequences, the nucleotides from the rail birds all pretty much matched up. Next week I will not be seeing Dr. Kirchman since it will be spring break, but he hopes to take a closer look at the rail DNA while I am gone and start analyzing it.