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

Summary

This protocol describes the CTAB method for extracting high-quality DNA from mycobacteria, overcoming challenges posed by their tough, mycolic acid-rich cell walls. The process involves enzymatic digestion, cell lysis with CTAB, and DNA purification through organic extraction and ethanol precipitation. This method produces DNA suitable for molecular studies and is reliable for mycobacterial research.

Abstract

Efficient and reliable DNA extraction is a fundamental step in molecular biology, providing high-quality genetic material for downstream applications such as PCR, sequencing, and genomic analyses. However, the unique cell wall composition of mycobacteria, rich in lipids and mycolic acids, poses significant challenges for efficient DNA isolation. The cetyltrimethylammonium bromide (CTAB) method is widely employed due to its efficiency in extracting high-purity DNA from mycobacterial species, including both fast- and slow-growing strains. The technique involves enzymatic disruption of bacterial cells using lysozyme and proteinase K, followed by the lysis of cell components using CTAB, which selectively binds and forms a complex with polysaccharides and proteins. DNA purification and concentration are achieved by organic solvent extraction and ethanol precipitation. According to our data, the CTAB protocol yields high quantities of DNA (200 - 1000 ng/µL) with high quality and minimal contamination as proven by gel electrophoresis and NanoDrop analysis. The DNA extracted from different mycobacterial species is of high purity and is suitable for various molecular studies. This study demonstrates the CTAB method's procedural steps, highlighting its utility in overcoming the unique challenges associated with mycobacterial DNA extraction.

Introduction

Extracting high-quality DNA is a fundamental step in molecular biology, essential for downstream applications such as PCR, whole genome sequencing, and other genomic studies. For Mycobacterium, a genus that includes both pathogenic species, such as Mycobacterium tuberculosis (M.tuberculosis), and non-pathogenic environmental species like Mycobacterium smegmatis (M. smegmatis), DNA extraction can be challenging due to their thick, lipid-rich, and hydrophobic cell walls^1,2,3. This tough cell wall necessitates utilizing specialized lysis methods to disrupt the cells and effectively release genomic DNA.

The cetyltrimethylammonium bromide (CTAB) method has proven reliable for isolating high-quality DNA from various mycobacterial species^4,5. CTAB, a cationic surfactant, binds to polysaccharides and other cell components, facilitating their removal during the extraction process⁶. Even with difficult samples, the CTAB-based protocol ensures the efficient isolation of intact and pure DNA, particularly when combined with enzymatic lysis, heat treatment, and organic solvent extractions^7,8. The CTAB method has played a crucial role in efficient DNA extraction from mycobacterial species, specifically for research involving whole-genome sequencing, metagenomics, and molecular diagnostics, which require pure and high-quality DNA input material^9,10. Compared to other DNA extraction techniques such as bead-beating or commercial kits, the CTAB protocol presented here offers a distinct advantage in preserving DNA integrity, an essential factor for long-read sequencing and epigenetic applications. While earlier studies have reported lower DNA yields with CTAB relative to mechanical disruption methods, they also emphasize its ability to minimize DNA shearing and maintain DNA integrity^11,12. To address limitations in purity and yield, the current protocol has been optimized with reduced incubation times and improved consistency across diverse sample types. This refinement was supported by findings from Opperman et al., who demonstrated that CTAB-extracted DNA from Mycobacterium avium cultures was of sufficient concentration and quality for downstream applications such as 16S rRNA sequencing and line probe assays, further supporting the application of the CTAB protocol as a versatile and scalable tool for mycobacterial research, particularly in studies requiring intact genomic DNA for advanced molecular analyses¹³. Additionally, next-generation sequencing (NGS) generally requires 100-1000 ng of high-quality genomic DNA¹⁴ and in the case of long-read sequencing, such as PacBio sequencing, requires ≥5 µg of High-quality DNA¹⁵, thus when large amounts of high-quality and intact DNA are required, CTAB remains an effective method¹⁶.

Establishing efficient, consistent, and effective DNA extraction methodologies from tough-to-lyse microbes is necessary to ensure reproducibility and reliability across laboratories, particularly for strain identification and epidemiological studies^5,7. This protocol provides details for extracting whole-genome DNA from Mycobacteria using the CTAB method. The current protocol aims to enable researchers to achieve consistent and reliable results in their molecular biology workflows by addressing the pitfalls and overcoming the challenges posed by the distinctive mycobacterial cell wall¹⁷.

Protocol

The representative results were generated with the approval of the REC: BES ethics committee, Stellenbosch University (BES-2024-22504). The reagents and the equipment used are listed in the Table of Materials.

1. Preparation of mycobacterial culture suspension

NOTE: Depending on the species being examined, this step must be conducted in either a Biosafety level 2 (BSL-2) or a Biosafety level 3 (BSL-3) laboratory. The incubation time and conditions of the culture also depend on the mycobacterial species' growth requirements due to their distinct growth rates and physiological characteristics.

1. Prepare 5 mL or 8 mL of bacterial culture in either Middlebrook 7H9 liquid media or MGIT media, respectively, supplemented with OADC and incubate at 37 °C for the required period to allow bacterial growth. Slow-growing mycobacteria require 10-14 days, while fast-growing mycobacteria require approximately 2-5 days.
   NOTE: A suspension of mycobacterial cells can be prepared by scraping a loopful of colonies into sterile double-distilled water if the starting culture is from solid agar plates (e.g., Middlebrook 7H10 agar).
2. Pour the liquid culture into 15 mL tubes.
3. Heat-kill the culture at 80 °C for 1 h in an incubator.
4. In the case of Mycobacterium tuberculosis or other pathogenic species that require BSL-3, transfer the heat-killed cultures to the BSL-2 laboratory.

2. Cell wall digestion and DNA extraction

NOTE: Once the culture is heat-inactivated, the following steps can be safely carried out in a Biosafety level 2 (BSL-2) laboratory. If the goal is extraction for long-read sequencing or alternative applications requiring high molecular weight DNA, be sure to utilize wide-bore pipette tips, and at vortex steps, replace vortex with inversion or pipette mixing.

1. Centrifuge the 15 mL tube at 3220 x g for 15-30 min at room temperature (RT). Discard the clear supernatant (ensure all the media is removed).
2. Thoroughly resuspend the culture pellet in 300 µL of Tris-EDTA (TE) buffer.
3. Transfer the resuspended pellet into a 2 mL tube.
4. Add 100 µL of lysozyme (10 mg/mL) and mix by pipetting up and down 5 times, then gently tap the tube to ensure uniform dispersion.
5. Incubate the mixture at 37 °C on a rotary incubator overnight (~16 h; lower incubation time might lower the lysis efficiency and thus lead to low DNA yield).
6. The following day, prepare a mixture of 5 µL of proteinase K (10 mg/mL) and 70 µL of 10% SDS. For example, if there are five samples, prepare 350 µL of 10% SDS and 25 µL of Proteinase K. Mix by inverting the tube to avoid foam formation.
7. Add 75 µL of the mixture to each sample.
8. Mix by tapping and incubate at 65 °C for 10 min in an incubator or heating block, with intermittent mixing by inversion.
9. Add 100 µL of 5 M NaCl.
10. Add 100 µL of preheated CTAB/NaCl solution at 65 °C.
    NOTE: Heat the CTAB/NaCl solution to 65 °C before ensuring pipetting accuracy, considering the viscosity of the substance at RT.
11. Mix by tapping until the solution becomes milky.
12. Incubate at at 65 °C for 10 min in an incubator or heating block, with intermittent mixing by inversion.
13. Add an equal volume (~ 675 µL) of chloroform/isoamyl alcohol solution (24:1) to each sample and mix by tapping.
    NOTE: Perform this step in a fume hood as chloroform is toxic if inhaled and requires cautious handling.
14. Centrifuge the samples at 12000 x g for 10 min at room temperature.
15. Carefully aspirate 550-600 µL of the aqueous (top) phase into sterile 1.5 mL tubes and label them appropriately.
    NOTE: Be careful not to disturb the other layers. If any layers are inadvertently disrupted, centrifuge the tube again.

3. DNA precipitation and elution

1. Add an equal volume (550-600 µL) of ice-cold isopropanol and mix by inverting the tubes.
   NOTE: If the DNA concentration is high, white precipitation may form during mixing.
2. Incubate the tubes at -20 °C for 30 min to 1 h.
   NOTE: Longer incubation times can enhance the DNA precipitation¹⁸ but may also lead to the coprecipitation of salts. Thus, overnight incubation can be performed if a low DNA concentration is expected.
3. Centrifuge at the highest speed (~21130 x g) for 30 min at room temperature to pellet the insoluble DNA.
   NOTE: Ensure the front side of the 1.5 tubes faces inward in the centrifuge rotor. At this step, the DNA precipitates at the bottom and along the back of the tubes. This is important to avoid disturbing the possibly invisible pellet in the next step. A white precipitate or pellet may be visible if the DNA concentration is high. The absence of a pellet does not indicate a failed extraction; proceed with the remaining steps.
4. Aspirate the supernatant from the front side of the tube without disturbing the DNA pellet or decanting it quickly while ensuring the pellet remains undisturbed.
   NOTE: It is better to aspirate the supernatant if the starting culture is small and a low DNA concentration is expected.
5. Add 1000 µL of ice-cold 75% ethanol.
6. Mix by inverting the tubes several times.
7. Centrifuge at 12000 × g for 30 min (in the same orientation as in step 3).
8. Aspirate or decant all ethanol without disturbing the pellet.
   NOTE: Steps 5-8 may be repeated if high salt coprecipitation is anticipated.
9. Allow tubes to air dry at RT overnight or for at least 30 min.
10. Add 25-50 µL of TE buffer (pH 8) to each tube to resuspend the DNA.
    NOTE: The DNA can be resuspended in Tris (10 mM, pH 8) buffer or Nuclease-free water if downstream applications are sensitive to EDTA.
11. Allow the DNA pellet to resuspend overnight at 4 °C.
12. Store at 4 °C for 2-3 days before performing a quality control assessment.
13. Store at -80 °C for long-term.

4. DNA quantification and quality control

NOTE: DNA quality control is generally performed using a microvolume spectrophotometer and gel electrophoresis. A fluorescence-based quantification system can also be used to quantify extracted DNA.

1. Spectrophotometer method
   1. Measure the DNA concentration and determine the quality following the manufacturer's instructions.
      NOTE: A DNA sample ideally has a 260/280 ratio between 1.7 and 2.0. A value of 1.8 is usually considered pure for DNA. Values below this range indicate contamination by residual protein, guanidine, or other reagents that absorb strongly at or near 280 nm. A 260/230 ratio between 2.0-2.2 is ideal. If the ratio is lower than expected, this may indicate the presence of contaminants that absorb at 230 nm, such as EDTA, phenol, and carbohydrates. Check the absorbance graphs obtained for the sample as well.
2. Gel electrophoresis
   1. Perform agarose gel electrophoresis¹⁹ to assess DNA integrity and determine whether fragmentation or degradation has occurred.
   2. To visualize DNA under the UV gel documentation system, use a 1.0% agarose gel containing SYBR Safe dye. Run the gel at 80 V for 45 min.
      NOTE: The aim is to have clear, distinct, high-molecular-weight bands above the highest band of the DNA ladder. Use fresh 1× TAE buffer each time.

Results

This DNA extraction protocol using the CTAB method (Figure 1) was assessed across various mycobacterial species such as M. tuberculosis, M. abscessus, and M. chelonea. DNA was extracted and compared regarding yield, purity, and integrity of the extracted DNA across samples.

DNA concentration varied across the mycobacterial species and ranged between 190-600 ng/µL and >1000 ng/µL in some instances, indicating that CTAB extracts high-yi...

Discussion

Representative results demonstrate the utility of the CTAB method in extracting DNA from mycobacterial species and addressing the challenges associated with extracting DNA from these organisms. Mycobacteria are well known for their complex and lipid-rich cell walls². The current results highlight several critical aspects that contribute to the success of this technique as it delivers high-concentration, high-quality intact DNA that should be suitable for downstream applications such as PCR, s...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 mL pipette tips	Any supplier
10 µL pipette tips	Any supplier
10% Sodium Dodecyl Sulfate (SDS)	Merck (Sigma-Aldrich)	71736-100 ML
15 mL polypropylene tubes	Any supplier
2 ml tubes and 1.5 ml tubes	Any supplier
200 µl pipette tips	Any supplier
2-PROPANOL, BIOREAGENT, FOR MOLECULAR B&	Merck (Sigma-Aldrich)	I9516-1L	Store in -20 °C
37°C incubator	Any supplier
5 M NaCl	Merck (Sigma-Aldrich)	S6546 - 100 ML
65°C incubator	Any supplier
Agarose gel	Merck (Sigma-Aldrich)
Bio-Rad UV gel documentation system,	BioRad
Centrifuge (for 15 ml tubes oe 50 ml) and Microfuge (for 2 ml tubes)	Any supplier
CHLOROFORM FOR ANALYSIS EMPARTA® ACS	Merck (Sigma-Aldrich)	1070242500
Chloroform/isoamyl alcohol (24:1)			In a fume hood, mix 384 ml Chloroform and 16 ml Isoamyl alcohol
CTAB/NaCl solution	Merck (Sigma-Aldrich)	52370-100 G	Dissolve 4.1 g NaCl in 80 ml distilled water. While stirring, add 10 g CTAB. Heat solution in 65°C incubator. Adjust volume to 100 ml with distilled water. Store at room temperature for up to 6 months.
DNA 1 Kb ladder	Thermo Fisher Scientific
ETHYL ALCOHOL, PURE, 200 PROOF, FOR MOL&	Merck (Sigma-Aldrich)	E7023-500ML	Mix 75 ml Absolute ethanol and 25 ml distilled water
Lysozyme (10 mg/ml)	Merck (Sigma-Aldrich)	10837059001	Reconstitute lyophilized lysozyme (brought to room temperature) with distilled water to 10 mg/ml
Middlebrook 7H9 media	Merck (Sigma-Aldrich)	M0178	Follow the manufacturer's guidelines for preparing the liquid media
MIDDLEBROOK OADC ENRICHMENT 6 x 100 ml F	BD	212240
Mycobacteria Growth Indicator Tube (MGIT) media	BD	245122
NanoDrop system	Thermo Fisher Scientific
P10 pipette	Any supplier
P1000 pipette	Any supplier
P200 pipette	Any supplier
Proteinase K, recomb, PCR grd 2x250mg	Merck (Sigma-Aldrich)	3115801001	Reconstitute vial of lyophilised Proteinase K with distilled water to 10 mg/ml. Freeze aliquots in 2 ml tubes at -20 °C
SYBR-safe	Thermo Fisher Scientific
Tris/EDTA (TE) buffer pH 8.0	Merck (Sigma-Aldrich)	8890-OP or 382499997
Vortex	Any supplier