BISC209/F13: Assignment 209 Techniques

Summary of Theory behind Molecular Tools for Identification of Bacteria by 16s rRNA gene sequencing

Due prior to Lab 7.

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 .
  Describe the use of fluorescent signals (not 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.
• DNA sequencing by Illumina™ method
• Identification of bacterial species using the Maldi-TOF spectrophotometer and Biotyper software. (There is information about the MALDI-TOF Bio-typer in the background in lab 8).

For this assignment we want you to describe how each of these tools works. 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.

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. 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 MALDI-TOF works can also be delved into from some of the manufacturers of those machines web sites such as Bruker (| http://www.bruker.com/products/mass-spectrometry-and-separations/maldi-biotyper/overview.html)or ABI or from the background information in Lab 8 about the MALDI-TOF BioTyper you will use in lab this semester. There is a guide to ABI sequencers (used in Sanger sequencing) in .pdf form available in Resources in Sakai. 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 amplification and a sequencing reaction in chain termination (Sanger) sequencing. Another visualization of how an automatic sequencer works in Sanger sequencing can be found at | http://users.ugent.be/~avierstr/principles/seq.html. Your goal is to write a clear concise summary of how each of these technolgies work to help us identify microorganisms. 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 explain it 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.