Polymerase Chain Reactions and Other Realizations
A few things to note;
1) I have updated and will continue to update the literature page so if you feel my journals do not provide adequate proof of research, you can check out what I'm reading on that page.
2) I took some time and wrote the past few journals out by hand before posting them online. Honestly, I didn't feel much of a difference but it was a good chance I think to work things out by hand, make notes in the margins, and feel/see the writing process happening in my very own notebook, just like when I was younger and working from yellow paper to draft to the next version.
3) In this journal, I discuss a method of DNA manipulation called Polymerase Chain Reactions. If you would like to be further educated on this method, this video from Khan Academy may be helpful.
So I know it's been a little longer than usual. Remember how I discussed steadiness and balance in my last post? I haven't published anything since then mostly because I've been trying very hard to find this homeostasis, although I cannot say that I have achieved such. And I know it's wrong to think that I can only write a journal when I've got results or some kind of profound thought, because that's definitely not how this works.
But I guess maybe something I am starting to realize in this never ending quest for normalcy is that I should not be looking for a set formula to apply whenever things get out of balance. It's a mindset that should be versatile enough for all aspects of life. At the same time, though, I get the feeling that normalcy (although moderate) doesn't begin with indifference or a lack of fervent desire to change. Something pronounced, a deliberate and clear intention on achieving the goal of level judgement. I suppose one could also beg to differ that it is also possible to take small steps, moderating areas of one's life part by part. I'm definitely no expert on this so I guess I don't really have an opinion but the first method I discussed definitely reminds me of something I just learned in the lab last week: Polymerase Chain Reactions, or PCRs as I will furthermore refer to them.
In a nutshell, PCRs are a method used to amplify a certain part of any DNA sequence - almost like a magnifying glass. They can be used to identify certain genetic mutations and genome mapping. For my particular purposes, I am using PCRs to eliminate a certain gene from one of my worm populations. I believe I briefly mentioned ordering a couple worm strains from Minnesota in the past. So the delivery came in a couple weeks ago but what we noticed is that one of the strains, although we can see the glia well, has many other genomic irregularities which can compromise the validity of my own experiment. So now, the plan is to isolate worms which have a heterozygous version of each gene (meaning once mated with wild type males, half of the progeny would not have this gene). A struggle with this is that there isn't only one supplementary mutation, there are three. Specifically, cima-1, rab-3, and caspase (more of a manipulation that targets certain cells in order to ablate them).
My first step was to get rid of the caspase before too many of the cells died. This wasn't much of a struggle mostly because this phenotype was not integrated into the whole DNA sequence, but rather, was part of an extra chromosome. What made this elimination simple is that in order for a genetic characteristic to have a truly lasting and prominent effect on the population, it must be integrated. Incorporating an extra chromosome makes for unstable genetic matter so the chances that it is phenotypically expressed in the animal is not complete. That means it is possible to identify which animals have been spared (through a fluorescent microscope) and move those to another plate so that they may parent a new (non-caspase) population.
In terms of the rab-3 mutation, getting rid of this one was a little more troublesome. Here, we had to keep on crossing in wild type males to get continually heterozygous animals until progeny after progeny, the mutation was eliminated.
And last but most certainly not least, is the cima-1 mutation. Now, this little sequence of DNA and I have some issues. For one, fluorescence can't distinguish its positive progeny from its negative progeny. Secondly, it would ruin my project's integrity since cima-1 is a synthetic mutation implemented for a very specific experiment. SO, this is the problem I have been trekking away at for the past couple weeks. After much searching for an alternative, the simplest (although not simple) solution came down to PCR.
The way this works is that I first identify the location of the mutation. Then I created a 5' - 3' sequence to match the first 100 or so nucleotide base pairs and a 3' - 5' antagonist sequence to match the last 100 or so base pairs. That way, my concoction (as I like to think of it), creates a fence around the mutation so that my tag polymerase section - which is just a reciprocal sequence of the mutation so that they can bind to one another - is protected on both sides to prevent anything that can separate the two complimentary sequences. Something to take note of here is that when we make these DNA primers, we have to make sure that we have the most percentage possible of C and G base pairs (Cytosine and Guanine). This situation is considered ideal because it allows for the most hydrogen bonds (the strongest kind) between the original DNA strand and its primer.
The way this would help me to identify whether or not a worm contains the cima-1 mutation is simply, if the primer binds to the DNA strand. To achieve this result, however, is a bit tedious. I first have to create the primer beyond just figuring out which base pairs we require - as in, put all the proteins together into about 1,000 strands of the sequence (which I am hopefully learning how to do this week). Then, I will put everything into a small test tube and into a machine called the DNA engine. From this machine, I will receive a report of the substance levels and various bonding stats. Then, we will run the mix of DNA mutations on a round of gel electrophoresis to garner whatever information we can. The final step involves us sending the reports to a DNA reading/crispr possessing institute in New Jersey which within a couple of days confirms or denies the existence of cima-1 in each of the worms we have incorporated into our little concoction.
So getting back to the more intangible problem I began this post with - what I described reminds me of PCRs because the two seem to have a common individuality between mutations, and people. Every mutation gets its own primer, no one's arguing with that. And likewise, every person gets to have a specific method to their madness. With some worm strain mutations, PCRs work best. With others, its a matter of time and multiple generations until the mutation is eliminated... I think you get my point. And no, I'm not saying people are worms, but that they certainly do possess similar characteristics. You know, being manic and erratic is exhausting and not finding a constant homeostasis is frustrating but maybe my problem is that I haven't invested adequate time into experimenting. Perhaps the root cause of my frustration is that I don't take risks in trying different things, not that I have extreme tendencies. But then again, maybe it's both. I'll be sure to let you know once I find out.