This DNA has a sequence and I have every intention to find out what it is! (and other stories)

In my quest to amplify a region of the nitrite reductase (NiR) gene that I can then ligate into a plasmid for in vivo testing, I needed to sequence my portion of DNA for three main reasons:

1. I needed to make sure that the restriction sites I added with gene specific primers were added to either end of the sequence. These restriction sites will allow me to cut out my amplified piece of DNA that is cloned into a cloning vector.
2. I need to verify that the sequence of DNA I've been cloning is indeed from the NiR gene, especially since the DNA sequence has been marginally longer on my agarose gels than anticipated.
3. Previous data acquired from a database told me I should be able to get a restriction enzyme to cut somewhere in the middle of the DNA sequence I had cloned, but the restriction enzyme wouldn't cut in the middle (but it did cut on either side of the DNA sequence that I cloned into a vector, as the vector had that restriction site on either side of the multiple cloning site). Essentially, that restriction site didn't exist in my DNA sequence like it should have.

So, couple weeks ago, I performed a PCR cycle sequencing reaction to start the process of sequencing. The purpose of this sequencing reaction is to amplify a region of DNA with DNA bases that have special tags. These tags are read by a sequencing machine yielding a series of base signals like below:

The color coded lines refer to a single base category (either A, T, G or C) and denote the signal strength at that spot in the sequence. Based on the predominant signal at a spot in the sequence, the software makes a judgment call as to which base is present at that location. Often it is quite clear to the machine and software that a particular letter of DNA is certainly an A at a specific location because there is a single strong signal peak. Other times there may be several signal peaks at a single location because of a lower quality sequencing reaction (e.g. there is too much or too little DNA to be read properly), which make it harder to determine which DNA letter actually exists in the DNA sequence.

In order for the DNA to be read by the sequencing machine, it needs to be single stranded. This means we make up separate PCR reactions with only one primer, which means that the one primer in a PCR reaction will attach to the DNA and copy the DNA until it falls off. To make it easier on myself, I followed a standard protocol that sequences a section of DNA that has been cloned into a plasmid vector. This way there is a buffer of vector plasmid sequence on either side of my DNA sequence of interest, where the primers that pair with the vector plasmid would bind.

I performed my PCR reaction and prepared it for the sequencing machine and was about to load my DNA onto the DNA sequencing plate (which goes into the machine), when I was notified that the machine was down and I couldn't sequence that day or possibly for the rest of the week. It took me a week or more for my samples to get read, but they finally did get read and my sequences were good enough to give me some answers.

I use a special computer program that will align the DNA sequences to one another. Running multiple samples of the same sequence will allow the program to compare answers and yield a single, best result sequence. Interestingly enough, while my four samples all matched each other, there was a small region (about 20 bases) that did not match up with the database sequence for my gene. This may have been why my restriction site wouldn't cut--because it wasn't there. One of my labmates is going to sequence the same gene soon, so I will be able to compare my results with hers and determine whether the predicted sequence in the database needs to be updated.

I can then take the sequence from the first program (the results) and pop it into another program, to quickly find all of the restriction sites that are present.

Here I can see that my BamHI and NotI restriction sites are indeed at either end of my sequence, with the EcoRI sites just outside of the Bam and Not sites. Because the EcoRI sites are in the vector which I cloned my sequence with, I know that the Bam and Not sites are on either side of my sequence like they should be.

At present I have ligated this section of DNA (the piece of the NiR gene) with the other portion of the NiR plasmid, and will transform some bacteria today. If the plasmid worked and was successfully cloned into my cells, they bacteria will be able to grow on the plate laced with ampicillin. Here's hoping!