Introduction to the molecular biological characteristics of soil

One of the most difficult types of samples in our lab is the soil. In the process of developing kits for extracting microbial DNA and RNA from soil and conceiving methods, we need to collect and record the organic content, texture, pH and collection sites of various types of soil. These factors are closely related to the soil microbial content, and also affect the yield of DNA and RNA.

Below, I will introduce some key issues affecting soil DNA and RNA extraction. To find out more about the organic ingredients found in the soil, click here (soil microbiology)

Humic substances:

The biggest problem that plagues the extraction of soil DNA and RNA is the humus content (humic acid such as humic acid and fulvic acid). Humic acid is degraded by organic matter (residues such as plants, microorganisms, etc.) and causes the soil to have a black color (the molecular structure of the phenylhydrazine group) (1). The humic acid molecules all contain carboxyl groups and phenol groups, and their molecular weights vary. Intermolecular chelates sequester macromolecular multivalent cations, making them very beneficial for re-absorption of microorganisms and plants (2). For more details on humus, please click here

Because humic acid and fulvic acid are macromolecular hydrophilic anionic polymers like DNA and RNA, they are easily extracted together with nucleic acids in the final step. So it is best to remove them before final elution of the nucleic acids.

IRT Technology (Inhibitor Removal Technology):

Inhibitor removal technology is a patented technology used by MO BIO to remove polyphenylene phenolic humic substances from soil samples. The working principle of this technology is to dissolve macromolecules such as proteins by first changing the pH value. The nucleic acid is not precipitated during this process, thereby separating and removing the inhibitor. The technique is equally applicable to heme and stool samples in the blood, melanin in the skin, and dyes on blood-stained clothing in forensic samples.

Lysis of microorganisms:

Removing the inhibitor is the first step, and lysing the microbial cells in the soil is the second step. Otherwise, the soil microbial structure cannot be accurately described. Mechanical lysis can quickly and efficiently lyse bacteria and fungi in the soil. Our lab prefers the Vortex Genie 2 because it has advantages over powerful high-speed bead mills.

Doing metagenomics?

One of the biggest benefits of using a vortex (Vortex) is that it can produce larger molecular weight DNA by breaking the cells with a relatively gentle force for up to 10 minutes. Little heat is generated during low-speed rotation to avoid DNA and RNA degradation caused by excessive heat accumulation. The use of MO BIO's lysis buffer can reduce DNA and RNA damage to a greater extent and ensure the integrity of nucleic acid molecules. Lysis buffer is also part of the IRT technology. Metagenomics requires large molecular weight DNA, so Vortex is the best choice.

Save money?

The vortex is small and inexpensive, especially suitable for laboratories with limited funds, and is also particularly suitable for setting up temporary laboratories in the wild. The vortex is lightweight and can handle up to 24 samples at a time. MO BIO has developed a range of adapters (Vortex adapters) for use on Vortex Genie 2. to accommodate Tube tubes of all sizes.

Stronger cracking?

The vortex and our other solutions are sufficient to lyse most organisms, while some fungal spores require more intense lysis. For this type of sample, we recommend adding a heating step before the vortex. After the addition, incubate at 65 ° C ~ 70 ° C for 10-15min (4, 5). Another alternative is to repeatedly freeze and thaw before vortexing. These enhanced lytic feet can be used against hard spores and fungal cells.

How about a high speed bead mill?

High-speed bead mills are sometimes an effective means of dealing with hard organisms, but too high a rate can excessively shear DNA, and the heat generated can also accelerate RNA degradation. In our laboratory, no high-speed bead mills were used to contribute to DNA yield by gel electrophoresis. On the contrary, Nanodrop's spectrophotometer has a high reading at 260 nm, which means that the DNA is severely damaged. (Nucleic acid protein quantitation instrument). The viable bacteria were counted using plate culture and it was found that the results obtained using the vortex meter and Precellys were similar. However, the high-speed bead mill Precellys increases the release of plant and insect DNA, diluting the proportion of microbial DNA. It should be avoided as much as possible during the experiment.

to sum up:

In the following articles, the methods for extracting soil DNA and the recommendations and techniques for improving yield and purity when using the Powersoil DNA Kit and the RNA Powersoil Kit will be detailed.

references:

• A. Piccolo (2002). "The Supramolecular structure of humic substances. A novel understanding of humus chemistry and implications in soil science". Advances in Agronomy 75: 57–134.
• FJ Stevenson (1994). Humus Chemistry: Genesis, Composition, Reactions. John Wiley & Sons, New York
• Techniques in Microbial Ecology, Burlage, Robert S., Ronald Atlas, David Stahl, Gill Geesey, and Gary Sayler, editors. 1998. Oxford University Press, New York, Page 277
• Development of Quantitative Real-Time PCR Assays for Detection and Quantification of Surrogate Biological Warfare Agents in Building Debris and Leachate Pascal E. Saikaly, Morton A. Barlaz, and Francis L. de los Reyes III Applied and Environmental Microbiology, October 2007, p. 6557- 6565, Vol. 73, No. 20
• Evaluation of five commercial nucleic acid extraction kits for their ability to inactivate Bacillus anthracis spores and comparison of DNA yields from spores and spiked environmental samplesLeslie A. Dauphin, Benjamin D. MoserJournal of Microbiological Methods, Volume 76, Issue 1, January 2009, Pages 30 -37