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The CTAB method
Purpose
The CTAB method described is designed to yield microgram quantities of high molecular weight (MW) DNA suitable for genotyping. Note that while high molecular weight DNA is not essential for Illumina sequencing (which produces very short reads of ~100 bp, DNA for direct ONT sequencing (without PCR amplification) should ideally be >30kb average size.
Principle
Although DNA can be extracted from MTB bacilli by a variety of methods, with a range of complexity, the method described here yields high quality, large fragment DNA from a colony scrape off solid media, or from a liquid culture such as a MGIT tube. Using a combination of enzymatic digestion and organic partition, a loop of colonies picked from an LJ slope yield 0.1-50 µg of DNA. Following resuspension and heat-killing of the colonies, bacteria are digested first with lysozyme to breakdown the cell wall then with proteinase K, which has further action on the cell wall but, importantly, digests any enzymes released by the lysed bacterium, including DNases. MTBhas a lipid-rich cell wall, and so two rounds of detergent are used, first Sodium dodecyl sulphate (SDS) and then Cetyl trimethylammonium bromide (CTAB). These detergents have action on molecules with different charges thus affecting different cell wall components. EDTA is used to chelate Mg+2 and Ca+2 ions, inhibiting DNase activity; similarly, high salt concentrations inhibit DNA-enzyme binding. Finally, organic solvents are used to partition the DNA to an aqueous phase, leaving lipids and proteins in the organic phase. The aqueous phase is then concentrated using isopropanol, this concentrates the DNA and removes excess salt. Isopropanol is used in preference to ethanol as a lower volume for precipitation can be used (1:1 rather than 2:1). Yeast tRNA is used during the alcohol precipitation step as it is an effective co-precipitant to aid in recovery of small amounts of nucleic acids and improve the DNA yield.
Obtaining the required quantity and MW of DNA
Due to the properties of MTB, it can be difficult to obtain the desired quantity of DNA (~5-44 ng/µl) needed for ONT sequencing. Cultures can be obtained from either MGITs or LJ slopes and the highest yield of DNA was obtained approximately 2 weeks after MGIT flagging. Culturing for DNA extraction using Middlebrook 7H11 slopes is not recommended as it does not yield as high MW as MGIT or LJ slopes.
Typically from a loop of cells we obtain 10µl of DNA at 1-500ng/µl (the variation may reflect the strain, medium, or age of cells). If a single extraction from a loopful of cells, or of 400 µl of liquid culture does not provide enough DNA (~53.3 ng/µl), then it is recommended that multiple extractions per isolate be done, then the eluted DNA combined. Keeping the elution volume low (<15 µl) will also help to create high concentration DNA. If there is enough DNA (e.g. ~400 ng), but it is too diluted, then ethanol precipitation is recommended. Ethanol precipitation is beneficial for removal of salts etc. to improve DNA purity, which will improve sequencing efficiency and flow cell pore longevity.
A full protocol, modified for sequencing will be uploaded soon