Whole-Genome Deoxyribonucleic Acid Extraction from Mycobacterium Species via the Cetyltrimethylammonium Bromide Technique

Our research centers on the genetic characterization of mycobacterial species and the complex nature of mycobacterial diseases in human. We place particular emphasis on nontuberculosis mycobacteria and their interactions with macrophage during coinfection with other species, especially microbacteria tuberculosis. Additionally, we focus on the speciation of pathogenic mycobacteria, identification on virulence-associated genes, and the single nucleotide polymorphism linked to drug resistance.

Due to the thick cell wall, lipidic and hydrophobic cell walls of mycobacteria DNA extraction has been challenging and requires specialized lysis techniques to break them down and efficiently release the genomic DNA. In our protocol, we are addressing DNA extraction from mycobacteria species using the CTAB method as a robust and effective method to produce high-quality DNA that is suitable for all genome sequencing and other molecular techniques. To begin, heat kill the liquid culture taken in a 15-milliliter tube at 80 degrees Celsius for one hour.

Then place the 15 milliliter tube in the centrifuge and spin at 3, 220 G for 15-30 minutes at room temperature. Using a sterile pipette, discard the clear supernatant, ensuring that all the media is removed. Resuspend the culture pellet thoroughly in 300 microliters of Tris-EDTA buffer, and transfer the resuspended pellet into a 2-milliliter tube.

Now, add 100 microliters of lysozyme solution at a concentration of 10 milligrams per milliliter to the tube and mix by pipetting up and down five times. Tap the tube gently to ensure uniform dispersion and place the tube in a rotary incubator at 37 degrees Celsius and incubate overnight. The following day, prepare a mixture of 5 microliters of proteinase K at a concentration of 10 milligrams per milliliter and 70 microliters of 10%SDS.

Add 75 microliters of this mixture to each sample. Mix the sample by tapping the tube and incubate at 65 degrees Celsius for 10 minutes, intermittently inverting or tapping the tube to mix. Next, add 100 microliters of 5 molar sodium chloride to the sample, followed by 100 microliters of preheated CTAB sodium chloride solution preheated to a temperature of 65 degrees Celsius to the sample.

Mix the sample by tapping until the solution becomes milky. Place the tube in an incubator at 65 degrees Celsius for 10 minutes, intermittently inverting or tapping the tube to mix. Now, add an equal volume, approximately 675 microliters of chloroform isoamyl alcohol solution in a ratio of 24:1 to the sample and mix by tapping.

Place the sample in a centrifuge and spin at 12, 000 G for 10 minutes at room temperature. Then, using a pipette, carefully aspirate between 550-600 microliters of the aqueous top phase into sterile 1.5-milliliter tubes and label them appropriately. Add 550-600 microliters of ice cold isopropanol to each tube and mix the contents by inverting the tube several times.

Place the tube in a freezer set to 20 degrees Celsius and incubate for 30 minutes to one hour. Next, centrifuge the tube at the highest speed, approximately 21, 130 G for 30 minutes at room temperature to pellet the insoluble DNA. Using a pipette, aspirate the supernatant from the front side of the tube without disturbing the DNA pellet.

Now, add 1, 000 microliters of ice cold 75%ethanol to the pellet, and mix the sample by inverting the tube several times. Centrifuge the sample at 12, 000 G for 30 minutes in the same orientation as previously used. After centrifugation, aspirate or decant all ethanol without disturbing the pellet.

Allow the tube to air dry at room temperature overnight or for at least 30 minutes. Then, add between 25-50 microliters of Tris-EDTA buffer at pH 8 to resuspend the DNA, and place the tube at 4 degrees Celsius overnight. After quality control assessment, place the tube in a freezer set to 80 degrees Celsius for long-term storage.

DNA yield obtained using the CTAB extraction method ranged between 190 nanograms per microliter and 600 nanograms per microliter. The 260/280 absorbance ratio ranged between 1.9 and 2.0, and the 260/230 ratio ranged between 1.8 and 2.2, indicating high purity DNA. A single absorbance peak was detected at 260 nanometers.

Agarose gel electrophoresis of high quality samples showed a distinct intact high molecular weight DNA band with minimal degradation. In some nontuberculosis mycobacterium species, DNA yield was less than 50 nanograms per microliter and purity ratios fell below the ideal range, indicating contamination.