This week, I was ready to begin my project in earnest and move quickly into the next phase, which will deal primarily with polymerase-chain reaction (PCR) primer design for application to multiple bacterial species. What the heck is that, you say?
Well, for you new readers, the gist of my project is as follows: When a lab has a sample of unknown DNA, it can begin the identification of that DNA by running it through a thermo-cycler process known as a PCR. This process subjects the sample to repeated healing and cooling, which breaks down the DNA contained in the sample. A substance called a primer is added to the DNA sample before the PCR is run, and during the PCR cycle that primer binds to the DNA fragments and assists in replicating the DNA until there is a larger quantity of it. The resulting larger sample is then sequenced to determine what organism it originated from. Why is this important? Imagine, if you will, that a violent crime has been committed and a miniscule DNA sample, left behind by an unknown suspect, has been found at the crime scene. This sample is too small to be sequenced (checked for its unique series of genes, contained in every organism's DNA), so it is useless for identifying who the suspect is. But by adding the tiny DNA sample to the proper primer and subjecting it to the PCR process, we can create more of the DNA, sequence it, and potentially identify the culprit.
There are many different primers used in PCRs; in fact, each primer is specific to a particular organism because primers target and adhere to a certain section of genes, unique to that organism only, during the PCR. The means that the need to use the correct targeting primer during a PCR, without knowing the origin of the DNA sample you wish to add it to, can be a little like a roll of the dice in certain circumstances. If a primer could be developed that was universal, or able to be applied to multiple unknown organisms, it would greatly shorten the amount of time needed to identify an organism.
That's where my project comes in. I am looking for primers that can be universally applied to bacterial species. Because E. coli has had its entire genomic sequence mapped, and a particular section of genes that was found in E. coli has also been identified in other bacterial species, I hypothesized that there must be a primer that would work during a PCR for all bacteria. In order to prove my hypothesis, however, I first had to get a crash-course in molecular biology techniques.
As a result, previous semesters have been focused on learning, developing, and implementing those techniques. I had to not only learn the vocabulary of a new field, I had to learn DNA extraction, verification, and amplification procedures. And since a major goal of my project is to also identify protocols that could be adapted into an easy method that the average student could potentially do in a Microbiology class, I had to also simplify those procedures as much as possible while still retrieving a viable, undamaged DNA sample.
That's what I have been doing, in a nutshell. The entire project has kept me fairly busy, but like I said this week I was ready to move forward. I did, in fact, manage to significantly expand my project beyond E. coli to include seven additional bacterial species, and I have begun the process of developing a primer that will target the specific genetic sequence contained in the DNA of those bacteria. Exact details of those developments to follow in a later blog.
Until then, have a most excellent week. Enjoy this pic of some human/chimpanzee gene sequences. Notice the similarities?
www.panspermia.org
"Multiple sequence alignment of the gene sequence of the human gene CLLU1 and similar nucleotide sequences from the syntenic location in chimp and macaque.
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