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 ly....

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 ch....

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