Chapter 2
DNA Isolation from Mycobacteria
Heena Jagatia and Daire Cantillon
Abstract
A vast array of molecular biology tools have been developed to investigate the Mycobacterium tuberculosis
genome since the advent of its successful sequencing in 1998. These tools, such as quantitative and end
point polymerase chain reaction, chromatin immunoprecipitation, and whole genome sequencing, require
genomic DNA extracted from lysed mycobacteria. There are numerous methods described in the literature
using mechanical, enzymatic, or chemical means to lyse cells and extract genomic DNA to varying degrees
of purity. Here, we describe appropriate methods for genomic DNA isolation from solid or liquid cultures
from both M. tuberculosis and nontuberculous mycobacteria.
Key words DNA, Precipitations, Quantification, Purification, Quantitative PCR, Chromatin immunoprecipitation, Next-generation sequencing, Standards
1 Introduction
The advent of DNA technologies and next-generation sequencing
(NGS) have allowed researchers to interrogate the genome of not
only Mycobacterium tuberculosis but also the genomes of nontuberculous mycobacteria (NTM) such as Mycobacterium abscessus and
Mycobacterium avium complex. Most molecular biology techniques are underpinned by the method of DNA isolation. However,
the lipid-rich and thick peptidoglycan mycobacterial cell wall is
resistant to many lytic approaches and the biological hazard
M. tuberculosis poses and can limit the choice of methods for
DNA isolation [1, 2]. There are robust methods of DNA isolation
for mycobacteria that are widely used which take the sample type,
sensitivity, duration of extraction, and the cost into account. The
choice of extraction method is important to remove inhibitory
elements that can interfere with downstream processing such as
polymerase chain reaction (PCR) and to minimize damage to
the DNA.
Firstly, simply boiling M. tuberculosis in sterile water for up to
15 min has been reported to be sufficient for killing M. tuberculosis;
Tanya Parish and Anuradha Kumar (eds.), Mycobacteria Protocols, Methods in Molecular Biology, vol. 2314,
https://doi.org/10.1007/978-1-0716-1460-0_2, © Springer Science+Business Media, LLC, part of Springer Nature 2021
59
DNA Isolation from Mycobacteria
Heena Jagatia and Daire Cantillon
Abstract
A vast array of molecular biology tools have been developed to investigate the Mycobacterium tuberculosis
genome since the advent of its successful sequencing in 1998. These tools, such as quantitative and end
point polymerase chain reaction, chromatin immunoprecipitation, and whole genome sequencing, require
genomic DNA extracted from lysed mycobacteria. There are numerous methods described in the literature
using mechanical, enzymatic, or chemical means to lyse cells and extract genomic DNA to varying degrees
of purity. Here, we describe appropriate methods for genomic DNA isolation from solid or liquid cultures
from both M. tuberculosis and nontuberculous mycobacteria.
Key words DNA, Precipitations, Quantification, Purification, Quantitative PCR, Chromatin immunoprecipitation, Next-generation sequencing, Standards
1 Introduction
The advent of DNA technologies and next-generation sequencing
(NGS) have allowed researchers to interrogate the genome of not
only Mycobacterium tuberculosis but also the genomes of nontuberculous mycobacteria (NTM) such as Mycobacterium abscessus and
Mycobacterium avium complex. Most molecular biology techniques are underpinned by the method of DNA isolation. However,
the lipid-rich and thick peptidoglycan mycobacterial cell wall is
resistant to many lytic approaches and the biological hazard
M. tuberculosis poses and can limit the choice of methods for
DNA isolation [1, 2]. There are robust methods of DNA isolation
for mycobacteria that are widely used which take the sample type,
sensitivity, duration of extraction, and the cost into account. The
choice of extraction method is important to remove inhibitory
elements that can interfere with downstream processing such as
polymerase chain reaction (PCR) and to minimize damage to
the DNA.
Firstly, simply boiling M. tuberculosis in sterile water for up to
15 min has been reported to be sufficient for killing M. tuberculosis;
Tanya Parish and Anuradha Kumar (eds.), Mycobacteria Protocols, Methods in Molecular Biology, vol. 2314,
https://doi.org/10.1007/978-1-0716-1460-0_2, © Springer Science+Business Media, LLC, part of Springer Nature 2021
59