Week 12-Semester 3. Finally, PCR results.

Hello! Welcome back to my blog.

This week, I was able to get lab time on the thermocycler, and thus began the final phase of my project. This last portion involves testing the nucleotide primers that I have designed for universality against the nine bacterial species selected for this project. I have already successfully tested a number of Universal Rice Primers against my extraction samples, so all that remains is to test these new primers in various combinations and at a number of different annealing temperatures and then analyze the data that provides.

Theoretically, the series of primers I am testing should successfully amplify DNA in three of my samples because the nucleotide sequences they contain were developed using the genetic sequences of those three bacteria. Therefore, I ran the first PCR of this project phase on the species S. enterica, S. sonnei, and K. oxytoca. I used an annealing temperature of 64°C during the reaction, which was squarely between my optimal primer temperatures of 62.4°C and 68.7°C. The results? DNA! However, the clarity of banding was extremely poor for S. sonnei and K. oxytoca. This may have been an indication of a number of things, including the amount of DNA present in my original extraction sample, the annealing temperature used, or the primers themselves.

In order to rule out/identify my sample itself as the potential culprit, I ran another PCR reaction using DNA samples, from a different extraction date, that were previously amplified using Universal Rice Primers. All controls in the reaction were an exact duplicate of the first PCR I ran this week.

The results? DNA, clearly visible for all three bacterial species, in my post-PCR gel.

This is a success for me on two fronts. One, it proved my hypothesis that my sample was responsible for the results of experiment #1. Two, it showed that the primers I designed work. This makes me quite happy, for it is an affirmation that my rudimentary understanding of genetics is improving.

Next week, due to these promising results, I will expand my PCR reactions to include the remaining organisms in this study. Until then, enjoy yourselves..and enjoy the following information about Neanderthal DNA that was recently extracted from an Italian specimen.

http://www.livescience.com/50458-oldest-neanderthal-dna-found.html

The above links to background information about Altamura man, a specimen of Homo neanderthalensis found in a cave in Italy approximately 150,000 years after his death.

This image shows the stalactites covering the specimen. Source: earth66.com 

Week 11-Semester 3

Hello! And welcome back.

There has been an ongoing issue these last couple of weeks with getting access to the thermocycler, so any plans to proceed with my project are on hold until that runs its course. My project has progressed to the point where all I need to do is collect data from PCRs so that I can evaluate the various primers I am using to amplify DNA; without the ability to run PCRs, all I can do is repeat what I have already done with extractions and basic lab methodology. Therefore, I have no data or conclusions to report this week.

This is actually a propitious development, as we are now nearing the last four weeks of the semester, the time when we all get slammed by course work. So although I am saddened that I cannot run reactions this week, I have instead been able to re-direct that time towards catching up assignments. This extra time is also providing a bit of relief for me because this has been my busiest semester to-date. Between the various jobs I have and the demands of academia, I have had about three days off total this entire semester. But lest one think that I am complaining, let it be known that I am grateful to be able to go to school and grateful to be in the S-STEM program.

That's it until I have some data to report. Have an Excellent Week!

P.S. Here is a picture for you, just because puppies always make people happy:

Photo credit: www.hdwallpapersinn.com

 

Week 10-Semester 3

Hello! And welcome back to my blog.

This week, I spent a great deal of time refining my extraction protocol to account for the increased growth encountered after I switched mediums from luria broth to TSB. I adjusted the initial sample size used to create a pellet for the extraction from 4.5 mL to 0.5 mL, and and reduced the amount of time that I centrifuged and vortexed each sample during the protocol application by 50% or more. This resulted in viable DNA samples for five of my eight bacterial species; the remaining three did not produce DNA. Because of the late date of the semester and the fact that I need additional data before I can write my research paper, I have decided to switch back to the growth medium I was previously using successfully for all eight species. This will allow me to get the data I need without losing any more time tinkering around with a basic procedure.

I also was able to spend some time running a PCR on one of the primer sets that I previously designed and have been excitedly waiting to test. Unfortunately, and to my profound disappointment, I have decided that the data generated from that experiment is unusable due to an error in the protocol used to provide the DNA sample used in the reaction.

I am going to re-test the reaction using another set of DNA samples that have already been evaluated for DNA using an electrophoresis gel. I have been storing the samples on ice for just such an occasion as this. The samples have been repeatedly subjected to gels, and have showed clear banding every time for all eight target species.

Sometime next week, I will set up a reaction using those samples and the primers I have designed. In the interim, please have an excellent weekend.

Please enjoy this photo of Proteus mirabilis, one of the species I am using. This little guy is commonly found in the human gastrointestinal tract, soil, and water. It can cause a number of infections in humans, and has been linked to the formation of kidney stones. (Davis & Zuber)

Photo credit:  CDC Public Health Image Library. CDC (PHIL #6691), 1976.
Link for more information from Davis & Zuber:  http://www.clinicianreviews.com/specialty-focus/nephrology/article/iproteus-mirabilisi-isolating-a-cause-of-kidney-stones.html

Week 9-Semester 3

Hello! Welcome back.

This week, I resumed the experimentation phase of my project after a brief hiatus caused by the thermocycler being unavailable. Without that essential piece of equipment, I have been unable to conduct any experiments, as the particular phase I am at within my project requires regular polymerase-chain reactions (PCRs) to test my hypothesis regarding universal primers. I did tinker around with some DNA extractions in the interim, as I am using some new base organisms for the project.

I experienced some minor hiccups in the extraction process of the new species, which after some review I have decided is a result of my switch to a new growth medium for my bacteria. To date, I had been using luria broth for cultures; with this new round of extractions, I switched to TSB. This richer medium is causing me to experience significant increases in culture growth during my standard 24-hour incubation period, which is resulting in larger pellet sizes upon centrifugation. These larger pellets were presenting some difficulty during the re-suspension step, and the enhanced vortexing required to properly mix the solution potentially resulted in an unusable extraction sample. After precipitation of the DNA was attempted using isopropanol, I had samples that were viscous and would not pellet. This caused me to not be able to retrieve a clean sample from the supernatant, and the extractions had to be discarded.

I repeated the process and adjusted the centrifuging times to account for the growth increase, which produced a less-dense sample pellet that was more easily re-suspended in solution. As a result, I was able to cleanly precipitate and isolate my DNA samples from the added bacterial species.

I then was able to proceed to the next phase of my project, which is the testing of nucleotide primers that I designed using genetic maps of the 16s ribosomal sequences of three of my target organisms. As you can imagine, I was extremely excited to finally get to this phase of the project.

I will post the results of this exciting new experiment on my very next blog, as I am still reviewing the data generated by the reaction. Until then, have a most excellent week!

Please enjoy the following abstract about the importance of 16s ribosomal gene sequencing; please download the full paper if you are so inclined:

http://www.ncbi.nlm.nih.gov/pubmed/18828852

And this:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC523561/

Plus, enjoy this representation of an enzyme used for PCRs:

Source: www.biochem.arizona.edu; DNA taq polymerase enzyme structure

Week 8-Semester 3

Hello, and welcome back.

Spring break is upon us, so unfortunately this means that no lab time will be logged this week. Yet, a blog is still due...so this week, in lieu of any experiments you will find here the abstract I submitted to the Estrella Mountain Student Conference. Cheers!

16s ribosomal gene sequencing is a standard technique for identification of bacterial species in the laboratory (Janda & Abbot 2007). Preparation of a bacterial sample for genomic sequencing is initiated by extracting DNA from the target species and conducting a polymerase-chain reaction (PCR) to amplify the DNA into a quantity sufficient for the sequencing process (Mao et. al. 2012). Nucleotide primers currently in widespread use by laboratories are targeted at specific variations of the 16s ribosomal gene found conserved among different bacterial species and are used during the PCR process to amplify the DNA sample. However, certain primers known as Universal Rice Primers (URPs) have been found to have universal application during the PCR across multiple bacterial species (Eun, et. al. 2002). This study analyzed URPs 2F, 4R, and 9F for universal application against eight bacterial species commonly found in laboratories. The bacterial species utilized were Salmonella enterica, Shigella sonnei, Proteus vulgaris, Proteus mirabilis, Klebsiella oxytoca, Providencia stuartii, Serratia marcescens, and Escherichia coli. The study also identified six previously unknown primers for analysis of universal application across the eight bacterial species. The identified primers were PCAT-1f-2015, PCAT-1r-2015, PCAT-2f-2015, PCAT-2r1-2015, PCAT-2r2-2015, PCAT-3f-2015, PCAT-3r-2015, PCAT-4f-2015, and PCAT-4r-2015. The study determined viability of the designated primer groups for use in universal application during the PCR process to enable subsequent identification of the target organisms.

BTW, here is an interesting link to a lay article about cloning the extinct mammoth.

 http://www.cbc.ca/news/technology/woolly-mammoth-cloning-attempt-revives-ethical-debate-1.2867654

And an image from the same article @ www.cbc.ca:

Week 7-Semester 3

Hello!

This week, I do not have an exciting development or even reams of data to report regarding my primers project. Due to a scheduling conflict with the BIO 181 labs, the thermocycler is unavailable for usage this week. Therefore, the next phase of my project is on hold until after the return from break.

This is particularly unfortunate, as I have been quite interested in testing my primers against the genetic sequences of my target organisms. As you may recall, I have already analyzed certain Universal Rice Primers (URPs) and found them to be not truly universal in application against the selected species under the controls I selected; the next phase is to examine whether or not the primers of my own design fare any better using those same controls. The essence of my project is the search for primers that can be used universally to amplify common bacterial species during a polymerase-chain reaction. This has gotten me a bit excited, as you may imagine, so having to wait another two weeks for results is stoking an excited anticipation that is almost untenable.

Instead of conducting experiments, I have been focused this week on expanding my project to include additional bacterial species. Welcome, Pseudomonas aeruginosa, to the fold. Also, in lieu of thermocycler time, I cultured fresh stock of the species I am analyzing and conducted DNA extractions on them. I banked them for later analysis. This, in a nutshell, was the progress I made this week.

Tomorrow brings the first field trip of the semester for our S-STEM group. We will be heading to the Phoenix Zoo for a little rest and relaxation, along with (hopefully) a little learning about the zoo's conservation efforts.

Until the semester resumes, be good to yourself.

PS: Here are some links to information about the black-footed ferret population recovery program, of which the Phoenix Zoo is a participating member. This only native North American ferret species was thought to be extinct until a lone colony was discovered in Wyoming in 1981. Since then, hundreds of kits have been bred and successfully introduced into the wild, including here in Arizona.

http://www.blackfootedferret.org/captive-breeding

http://www.azgfd.gov/w_c/blackfooted_ferret.shtml

Week 6-Semester 3

Hello! Welcome back to my blog.

As mentioned last week, I have been spending some time perusing the 16s gene sequences of my target organisms and was able to design a number of primers for use in PCR. The primers have been ordered, and that portion of my research is in a holding pattern until the order arrives.

In the meantime, I have been testing Universal Rice Primers (URPs) for universality against the eight bacterial species I am studying. URPs are called universal arbitrary primers because, rather than targeting specific gene sequences without variation, they adhere to and amplify random nucleotide sequences during the PCR process. Theoretically, this allows them to be used with a greater variety of organisms than the sequence-specific primers I am testing. However, in my first attempt to amplify DNA using these primers and a protocol previously used by a former S-STEM student, my PCR was unsuccessful. I surmised that this was likely due to the lack of necessary data from the previous student's experiment, as all I had to go on when attempting replication of the experiment was the poster that had been created with a synopsis of the experiment. Detailed, relevant information, such as the concentration of the primer dilution used, was missing.

In order to surmount this obstacle, I devised an experiment that utilized repetition and manipulation of variables to potentially identify the reason(s) for the failure of the first experiment. Using the same DNA samples that were utilized in the first test, I changed the annealing temperature of the PCR protocol to a lower temperature than previously used; I also repeated the original primer dilution but added another dilution factor as a variable to test concentration effect on PCR results. Given the variables being tested, I had a total of forty-eight samples being tested in one experiment.

The results? DNA amplification successful! Data analysis indicated that primer concentration did affect PCR results, as there were variances in banding frequency and size when the electrophoresis gels were reviewed post-PCR. However, I determined that temperature was the primary factor for the differences in results between experiments #1 and #2; given that I repeated all of the original controls, changed only the annealing temperature, and left the original primer concentration intact for the second experiment, this indicated that temperature was the culprit in the first experiment.

The addition of a primer concentration variable to the experiment did provide valuable data. Gel analysis revealed that DNA banding was less prevalent at the lower concentration for all but one of the primers, which instead showed an increase in banding clarity. This result will help me in designing future experiments using these URPs, as it revealed the optimal dilution concentration formula for primers tested at the annealing temperature selected.

Next week, I will be repeating the experiment using primers of my own design. Until then, in the words of S'chn T'gai Spock, "Live long and prosper."

And speaking of rice, here is an interesting video on GMO'd golden rice.