Introduction

During week seven we redeemed our previously failed attempts of PCR. The sample I used was labeled MW 00. We isolated genomic DNA from bacterial mat samples and amplified it with primers specific for all bacteria 16S ribosomal DNA. During week eight the PCR product was run on an agarose gel and during week nine we conducted DGGE analysis on the same product.

PCR

Results: The lane labeled C is the negative control. The lane labeled 1 has a 1:1 dilution of the sample. The lane labeled 10 has a 1:10 dilution of the sample and the lane labeled 100 has the 1:100 dilution. The negative control has no visible band, indicating that I did not contaminate it. All three dilutions produced bands at approximately the same location, as was expected from the primers used.

C 1 10 100

	DGGE
Methods: To produce the gradient needed in the gel, Liz and I mixed 12 ml of each parent solution (low and high), and the catalysts (APS and temed) into conical tubes. The high concentration tube also received 200 ul of Dcode dye. We then poured these solutions into the “gradient maker” without spilling (I was not successful with the not spilling part).
Methods: To pour the gel we opened both valves and allowed the two concentrations to slowly mix while draining through a plastic tube and into the washed and assembled plates. Once finished pouring, I inserted the comb and we allowed the gel to polymerize. We loaded the gel with our samples and loading dye mixed 1:1.
Results: Unfortunately, our gel ran for too long so the fragments did not remain in their unique locations in the gel as expected. If there were definite bands on this gel, each band would have represented a different member of the population.

Discussion

Denaturing Gradient Gel Electrophoresis (DGGE) separates sequences based on their nucleotide composition by “melting” the DNA using a chemical gradient. DNA melts at different temperatures, or chemical concentrations, based on its base pair composition. Higher percentages of guanine and cytosine in the sequence raise the melting temperature and therefore cause the sequence to travel farther down the gel before it is denatured. Denaturing causes the DNA fragment to physically stop moving through the gel because the single stranded regions get caught in gel. Every member of a population of bacteria has a unique base pair composition, and thus will give a distinct and unique band on the gel. Theoretically, each band can be excised from the gel and amplified using PCR.

As mentioned above, our DGGE gel ran for too long and as a result we did not obtain distinct bands. If the gel did have distinct bands on it, each band would have represented a different member of the population. This technique would have helped to assay the diversity of the red bacterial population in the original mat samples.

Practical Applications of DGGE

Application #1

Reference:

Kawai, Mako., et al. “16S Ribosomal DNA-Based Analysis of Bacterial Diversity

in Purified Water Used in Pharmaceutical Manufacturing Processes by

PCR and Denaturing Gel Electrophoresis.” Applied and Environmental Microbiology. 68.2 (2002): 699-704.

Summary

These researchers set out to analyze the purity of the water used in the drug making process. To do so, they collected and filtered water samples from the production facility to isolate any bacteria. They then plated these bacteria on standard media to assay the diversity of the population. In addition, they analyzed the isolated bacteria by extracting the DNA, amplifying it using PCR techniques and running it through DGGE. The researchers then isolated the bands from the DGGE gel, amplified each band individually and sequenced the DNA. Their results indicated that bacteria not identifiable by standard culture procedures were present in the water, suggesting that quality control measures dependent on culture techniques may not be sufficient for identifying the presence of bacterial contaminants. In addition, the sequences revealed that the dominant bacterial species in the water samples belonged to Alphaproteobacteria.

Application #2

Reference:

Wang, XinJing, MD, PhD, et al. “Mutation in Gene Responsible for Cystic

Fibrosis and Predispostition to Chronic Rhinosinusitis in the General

Population.” The Journal of the American Medical Association. 284.14

(2000): 1814-1819.

Summary:

These researchers set out to determine whether mutations in the gene responsible for causing cystic fibrosis predispose the carrier to Chronic rhinosinusitis (CRS). To do so, the doctors assembled a group of individuals who met the clinical diagnosis criteria for CRS and a control group of individuals who had no symptoms of the disease. DNA samples from all study participants were screened for the sixteen most common mutations in the gene. In order to find other less common mutations that may have the same effect, the researchers ran the DNA samples through DGGE. DNA samples that showed an “abnormal migration pattern” were recovered from the gel and sequenced. This allowed the researchers to quickly identify patient samples that had mutated genes without requiring them to sequence the DNA of all participants. From their analyses, the researchers concluded that mutations in the gene responsible for CFG “may be associated with the development of CRS in the general population.”