291787 Developing a Protocol to Concentrate and Isolate DNA From Deep Sea Sediment Samples

Monday, October 29, 2012
Hall B (Convention Center )
Daniel T. Speckhard, Chemical Engineering, Stanford University, Stanford, CA

Chloroflexus bacteria, Dehalococcoides, have been extensively studied in the past ten years. Dehalococcoides bacteria have shown to reducitvely dehalogenate common groundwater pollutants like trichloroethene (TCE) and tetrachloroethene (PCE). In order to better understand the metabolic pathways of Dehalococcoides, engineers have been keen to study Dehalococcoides living in deep-sea environments. These deep-sea Dehalococcoides bacteria grow in environments where polychlorinated compounds are not present in significant concentrations. Understanding the metabolic pathways of Dehalococcoides in environments without artificial pollutants could help engineers better understand the dehalogenation pathways in order to optimize the process of removing TCE and PCE from groundwater. Thus, the ability to study the DNA of deep-sea Dehalococcoides bacteria could help scientists provide insight into the genetic evolution of Dahalococcoides and its production of crucial enzymes to reductive dehalogenation encoded by the rdh gene.

Unfortunately, samples from deep-sea sediment have very low DNA concentrations. Moreover, these samples like most sediment samples have very high humic acid concentrations that act as inhibitors for enzymatic amplifications like PCR.  Therefore, in order to perform PCR, the humic acids must be eliminated and the DNA must be concentrated. There are several common methods available to scientists and engineers to perform this concentration and isolation of nucleic acids, such as the use of magnetic beads or simple electrophoresis on an agarose gel. These methods work well except for samples where the DNA concentrations are extremely low. In such low DNA concentration cases the losses effected in these operations are too high for engineers to use these methods to prepare PCR samples.

Boreal Genomics, a Canadian company has developed a machine, the Aurora Nucleic Acid System to concentrate nucleic acids through the use of complex electrodynamics on an agarose-based gel in a reusable plastic cartridge. An electric potential is created from the left to the right of the cartridge that separate charged molecules. Upon entrance to an octagon shaped area in the gel, the negatively charged molecules feel the force of a heterodyned alternating applied voltage that causes molecules to concentrate in the center of the cartridge. What determines whether molecules will focus at the center of the cartridge is the proportion (k/D)^0.5 where k is an electromagnetic constant and D is a constant of diffusion for each molecule. DNA with its very heavy mass and high linear charge density overcomes the force to diffuse from the center and is concentrated in a 60µl well. Unfortunately, deep-sea sediment samples contain very high concentrations of salt, which increases the resistance of the gel and causes the gel to overheat and melt causing havoc in the concentration of DNA.

Mini-dialysis units with a permeable membrane to salts were used in order to reduce the salt concentrations of DNA containing high salt concentration samples. After about 100 minutes in the dialysis unit the percent removal of NaCl for a sea-salt DNA sample nears 95%. The DNA loss has been shown for the process to hover around 20%.

Experiments on the Boreal machine show that only nucleic acids of size 500bp and above are retained, which means genomic DNA from Dehalococcoides should not be a problem. DNA percent loss from the Boreal machine hovers above 50%. When coupled with the percent yield of DNA from the mini-dialysis units gives an overall yield of about 40%, which is on the low end of what is needed for enzymatic amplifications. The technology in the Boreal machine however has shown to be promising and renewed efforts to optimize a protocol for the machine seem to indicate that the yield can be improved to offer a new exciting technology for studying DNA from deep-sea sediment samples.

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