Summary of Theory behind Molecular Tools for Identification of Bacteria by 16s rRNA gene sequencing
Due at the beginning of Lab 7
Write a narrative description of the identification of your bacterial isolates as part of your experimental design. Begin with your investigative goals and how isolating and identifying a few selected bacteria in your community provides useful evidence in your investigation. Include a description of the theory behind the following techniques that we used to identify our bacterial species by molecular tools:
Polymerase chain amplification of the 16s rRNA gene,
DNA sequencing by chain termination, sometimes called Sanger sequencing (not 454 pyrosequencing).
Describe the newer use of fluorescent signals instead of the older radioactive labels. Include how an automatic sequencing machine separates the DNA fragments that result from a sequencing reaction and then how the machine reads the fluorescent signals from the ordered DNA fragments. Also include how bacteria is identified from this 16s rRNA gene sequencing information from a public data base like RDP or NCBI Blast.
NOTE: WRITE THIS AS A SINGLE NARRATIVE (including required elements in whatever order feels appropriate; include appropriate transitions); DO NOT STRUCTURE AS BULLETED POINTS AND BE THOROUGH! (This assignment should be a couple of pages not just a few sentences.)
You have used these molecular tools and described them as a Material and Methods section, but you haven't yet been required to explain the theory behind how they accomplish identifying unknown bacteria by genus and species name from DNA sequencing. One of the problems in using sophisticated molecular tools is that you can have a very successful lab day, yet it can be mostly "hands on, brain off". Since much of what you have been doing is pipeting, mixing, and incubating of miniscule quantities of liquid reagents that come in kits, it is easy to lose sight of what is actually happening in those tubes or spin columns at each stage. The problem of "doing without knowing" is exacerbated by kit manufacturers who make their reagents "proprietary". That prevents us from knowing exactly what's in them, making it even harder to follow the chemical or physical reactions.
Your goal here is to understand and describe how it all works. These tools were discovered by scientists who published their findings. You don't, however, probably need to go to primary literature (Sanger's original paper, for example) to find out how Sanger sequencing works. There are good animations of Sanger sequencing, pcr, etc. prepared by the Dolan DNA center at [| http://www.dnalc.org/resources/animations/]. Pay particular attention to the difference between a polymerase chain reaction and the Sanger sequencing reactions. Wikipedia is a great place to start to find out some background for what you need to know about polymerase chain reaction. Understanding how an automatic sequencing machine works can be delved into from some of the manufacturers of those machines web sites, such as ABI. A link to some information about how an automatic sequencer works can be found at | http://users.ugent.be/~avierstr/principles/seq.html. There is more information in guide to ABI sequencers in .pdf form available in Resources in Saki.
Although it won't be difficult to find out the principles behind Sanger sequencing and polymerase chain reaction and to understand why we picked the 16S rRNA gene for sequencing to differentiate our bacterial species, it will be challenging to condense it to essentials in this summary. Being able to distill and write for non-scientists so that they understand how it works but don't get lost in unimportant minutia will be important when you describe your experimental design in your final paper.
Another good source of information is the background information found in this wiki. Be careful about inadvertent plagiarism.
The goal of this assignment is to make sure that you have a clear understanding of the biological and chemical basis of these common molecular tools.