Principles of Denaturant Gradient Gel Electrophoresis (DGGE) Analysis: This is a fingerprinting methodology.

. In theory, one band represents one microo rganism. Band intensity might be correlated with abundance of the microorganism, but not necessarily! It is based on nucleic acid sequenceconsiders the content of guanine and cytosine . It is NOT based on size. The sample is a mix of DNA from different microorganisms. Fragments will have t he same size but different sequences. The fragment size should be between 300 600 base pairs. If the fragment is any larger, the fragment wont migrate. The denaturant agents used are urea and formamide. Use of RNA vs. DNA: o Cannot use RNA directly. Use it to make DNA. RNA only identifies viabl e (alive, active) microorganisms. RNA degrades easily. The profile can rapidly change during the sampling. o DNA identifies regardless if the microorganisms are viable or not. Must do PCR first. This increases the amount of DNA from the gene you are ampli fying. Amplify to generate enough copies of one gene from one microorganism so that it can be detected by DGGE. PCR first denatures the DNA. Then it uses pri mers to specify what sequences to amplify. It uses tags to put the compliments where needed. o 16S rDNA is the genetic marker used for prokaryotic organisms. o 18S rRNA is the genetic marker used for eukaryotic microorganisms. o Must have a GC clamp to avoid total denaturing. The clamp allows the DN A to partially unzip exposing the nucleic acid sequences. o Have a forward primer and a reverse primer. The GC clamp is attached to the forward primer. This generates a strong product that is not denatured duri ng the electrophoresis. The size of the GC clamp is 20 36 base pairs. Two types of DGGE gels: Perpendicular and parallel o Perpendicular gels help to decide the proper concentration of denaturant . Usually run from 0% to 70%. The electrophoresis travels top to bottom and th e denaturant gradient increases from left to right. o Both the electricity and the denaturant gradient flow in the same direct ion in a parallel gel. The electrophoresis travels top to bottom and the denatu rant gradient increases from top to bottom. The concentration for the denaturan t is usually 30% to 65% for all communities. SOP: Preparing 30% and 60% denaturant solutions for DGGE Virginia Tech Molecular Food Microbiology Laboratory- Monica Ponder, PI. Prepared by: Gabriela Lopez-Velasco Date: 12-22-2007 Materials needed: 40% Acrylamide/Bis (37.5:1) Bio-Rad Catalog # 161-0148 TAE buffer 50X Formamide (deionized) Fisher Catalog # BP 227-100 Urea Fisher Catalog # U15-500 Autoclaved and distillated water Spatula Top loading balance Glass Beaker To prepare the solutions see the following table and instructions NOTES: Denaturant solutions can be stored at 4C in a glass flask for up to 1 mont h. The flask is to be amber or covered with aluminum foil. Allow the solutions to warm to room temperature 1 hour before use When adding the reagents try to avoid the introduction of oxygen. Do not shake the solutions, and pour the solutions slowly. The 50 mL is enough solution for 3 gels

Table 1: Solutions used in preparation of 30% and 60% denaturant solutions for D GGE Solution 30% denaturant solution 60% denaturant solution Urea 6.3 g 12.6 g 40% Acrylamide/Bis (37.5:1) 10 mL 10 mL TAE 50X 1 mL 1 mL Formamide 6 mL 12 mL dH2O Enough to bring the solution to 50 mL Enough to bring the solution to 50 mL Instructions: Weigh the urea into two separate beakers using a top loading balance Using a pipette, add the appropriate amount of Acrylamide/Bis to each beaker Using pipettes, add the appropriate amount of TAE 50X buffer and formamide to ea ch beaker Add about 20 mL of water to each beaker Place a stir bar into each beaker and stir using a stir plate until the urea is in solution. The solutions will be cool. Do not warm the solutions. Using a graduated cylinder, add enough water to bring the solution to 50 mL.