Summary of Theory behind Molecular Tools for Identification of Bacteria by 16s rRNA gene sequencing
Due prior to Lab 7. Please submit an electronic copy to your dropbox and bring a hard copy to class.
Write 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 or illumina high throughput sequencing).
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.
- Identification of bacterial species from 16s rRNA gene sequencing information using a public data base like RDP or NCBI Blast.
- Identification of bacterial species using the Maldi-TOF spectrophotometer and Biotyper software. (You will need to read the background in lab 9 to summarize the Maldi-TOF Biotyper theory).
You have used or will use these molecular tools and will include them in a Material and Methods section, but for this assignment we want you to explain the theory behind how they accomplish identifying unknown bacteria by genus and species name from DNA sequencing or molecular fingerprints. 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 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 these techniques work. These tools were discovered by scientists who published their findings. You don't, however, need to go to primary literature (e.g. 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 a topic. Understanding how an automatic sequencing machine or the MALDI-TOF works can also 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 Sakai. Another good source of information is the background information found in this wiki. We recommend that you use the background found in Lab 9 where you will be introduced to the most current tool used to identify bacterial isolates in humans: The Maldi-TOF Biotyper. Read over information provided in Lab 9 and write a clear concise summary of the theory. Be very careful to avoid inadvertent plagiarism, while it is tempting to copy and paste from websites, you need to understand the theory yourself and then write in your own words.
Although it won't be difficult to find out the principles behind Sanger sequencing, the polymerase chain reaction and the other techniques or to understand why we picked the 16S rRNA gene for sequencing to differentiate our bacterial species, it will be challenging to condense your writing to the essentials for 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.
The goal of this assignment is to make sure that you can communicate a clear understanding of the biological and chemical basis of these common molecular tools as we applied them to our work this term.