Koala Poop Microbiome Class: Week 4, PCR

(This blog post was prepared by students enrolled in the Koala Poop Microbiome Class in the Fall of 2016 at UC Davis)

Polymerase Chain Reaction

Polymerase Chain Reaction (PCR) amplifies a small amount of DNA into a much larger amount of DNA within a short time.  The purpose of this lab was to prepare the DNA extraction samples from the previous week for PCR, which was relatively simple. Though not all of the DNA extractions were successful, every student had at least four tubes of DNA from different isolated bacterial colonies to work with. The first step was to label the PCR strip tubes using a numbering scheme. We next added Master Mix and extracted DNA to its corresponding tube. Along with our bacterial DNA samples we included positive and negative control tubes to ensure the results of the PCR were accurate. The positive control consisted of a known bacterium’s DNA while the negative control was just the water that was used to make the master mix.

The Master Mix

What is it for: The master mix contains the nucleotides, primers, and heat resistant polymerase that are essential to the replication and amplification of the desired gene sequence.

What are the primers: Universal primers for bacterial 16S rRNA are present in the master mix. The 16S rRNA region is as unique as a fingerprint with small mutations between species that allow us to identify a bacterium based on its specific sequence.

-FUN FACT: If we were trying to identify a fungus we would amplify the 18S region of rRNA and if we were looking at a nematode’s genome we would amplify the 23S region!!

 

-This 4 minute video really helps make sense of the PCR process. It was produced in the Institute of General Organic Chemistry of the CSIC by Guillermo Corrales, to help promote Science Communication: https://www.youtube.com/watch?v=iQsu3Kz9NYo

 

PCR allows scientists to take a small fragment of DNA and (under optimal conditions) replicate it exponentially to a useful amount for testing and identification. It is a very common process and is extremely valuable in many fields of the science community; such as, diagnosing diseases, identifying bacteria, matching criminal DNA to crime scenes, performing paternity tests, and many others. It is a relatively simple procedure that has numerous applications in the vast world of science.

For many of us students, learning the in’s and out’s of PCR has allowed us to better familiarize ourselves with various lab techniques and proper lab etiquette. The ability to carry out a basic PCR will come in handy and be of great value if we choose to pursue other undergraduate research studies.

In correlation with our current project, PCR is the transition point from laboratory work to computer work. This technique amplifies the 16S rRNA gene of the different bacteria allowing us to sequence the genes and determine the identities of these gut bacteria that we isolated from Koala fecal samples on day 1. Prior to using sequencing software though, we will run samples of our amplified rRNA through gel electrophoresis. Next week, we will be making gels!

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David Coil

David Coil is a Project Scientist in the lab of Jonathan Eisen at UC Davis. David works at the intersection between research, education, and outreach in the areas of the microbiology of the built environment, microbial ecology, and bacterial genomics. Twitter