however, most containment level 3 (CL3) standards of practice
(SOP) require a minimum of 2 h to ensure complete killing
[3]. Others include beads and sonication followed by further purification steps with proteinase K and phenol/chloroform to the
boiling method [4, 5]. Secondly, for high DNA purity, the cetyltrimethylammonium bromide (CTAB) method has been used,
which briefly involves a proteinase K treatment, CTAB, and NaCl
to precipitate proteins followed by a chloroform–isoamyl alcohol
extraction [1, 2]. Finally, a commercial kit can be used to filter and
concentrate the nucleic acids for which the quality and quantity are
assessed by NanoDrop and/or other fluorometric quantification
assays. The quality of the DNA is crucial for a range of applications,
for example, whole genome sequencing, chromatin immunoprecipitation sequencing (ChIP-seq), and quantitative PCRs, while it
is not so important for colony PCR.
Since the DNA isolation chapter in the second edition of
Mycobacteria protocols [6], there have been significant advancements in the tuberculosis field for DNA extractions. Here we
describe several DNA isolation approaches for a variation of applications: isolation of plasmid DNA for colony PCR in
M. tuberculosis, isolation of DNA from ChIP in M. tuberculosis,
extraction of genomic DNA for qPCR standards from
M. tuberculosis, and isolation of DNA from M. tuberculosis and
NTM for NGS.
1.1 Isolation of DNA
from M. tuberculosis
for Detection of
Plasmid Inserts
As discussed, there are many methods that can be used to extract
DNA; however, some methods do not completely inactivate
M. tuberculosis and using boiled lysate may carry over inhibitors
for downstream applications [1, 7, 8]. The cost of enzymatic lysis
and detergents or commercial kits can be expensive. To lower the
cost and risk of a laboratory acquired infection, we describe an
inexpensive method to inactivate and isolate genomic DNA from
M. tuberculosis ready for detection using PCR.
1.2 DNA Isolation
Using ChIP in M.
tuberculosis
The study of protein–DNA interactions has been applied using
electrophoretic mobility shift assays (EMSAs), DNase I footprinting, and ChIP [9, 10]. The advantage of ChIP over the other
available techniques is that we can study the protein–DNA interactions in vivo [11]. ChIP (and ChIP-seq) has been used in
M. tuberculosis thus far for investigating transcription factor binding [12–14] and virulence factor binding [15, 16]. Others have
used advanced ChIP techniques such as ChIP-on-chip which
entails ChIP and hybridization to microarrays to study regulons
[14, 17]. Additionally, ChIP-seq has been used to complement
other techniques such as RNA sequencing to understand regulons
[18]. Here, we focus on the application of ChIP in M. tuberculosis
which can be used to study the binding patterns of a single and/or
multiple proteins to the genome such as transcription factor, transcription cofactors, histone modifications, and DNA repair
60
Heena Jagatia and Daire Cantillon
(SOP) require a minimum of 2 h to ensure complete killing
[3]. Others include beads and sonication followed by further purification steps with proteinase K and phenol/chloroform to the
boiling method [4, 5]. Secondly, for high DNA purity, the cetyltrimethylammonium bromide (CTAB) method has been used,
which briefly involves a proteinase K treatment, CTAB, and NaCl
to precipitate proteins followed by a chloroform–isoamyl alcohol
extraction [1, 2]. Finally, a commercial kit can be used to filter and
concentrate the nucleic acids for which the quality and quantity are
assessed by NanoDrop and/or other fluorometric quantification
assays. The quality of the DNA is crucial for a range of applications,
for example, whole genome sequencing, chromatin immunoprecipitation sequencing (ChIP-seq), and quantitative PCRs, while it
is not so important for colony PCR.
Since the DNA isolation chapter in the second edition of
Mycobacteria protocols [6], there have been significant advancements in the tuberculosis field for DNA extractions. Here we
describe several DNA isolation approaches for a variation of applications: isolation of plasmid DNA for colony PCR in
M. tuberculosis, isolation of DNA from ChIP in M. tuberculosis,
extraction of genomic DNA for qPCR standards from
M. tuberculosis, and isolation of DNA from M. tuberculosis and
NTM for NGS.
1.1 Isolation of DNA
from M. tuberculosis
for Detection of
Plasmid Inserts
As discussed, there are many methods that can be used to extract
DNA; however, some methods do not completely inactivate
M. tuberculosis and using boiled lysate may carry over inhibitors
for downstream applications [1, 7, 8]. The cost of enzymatic lysis
and detergents or commercial kits can be expensive. To lower the
cost and risk of a laboratory acquired infection, we describe an
inexpensive method to inactivate and isolate genomic DNA from
M. tuberculosis ready for detection using PCR.
1.2 DNA Isolation
Using ChIP in M.
tuberculosis
The study of protein–DNA interactions has been applied using
electrophoretic mobility shift assays (EMSAs), DNase I footprinting, and ChIP [9, 10]. The advantage of ChIP over the other
available techniques is that we can study the protein–DNA interactions in vivo [11]. ChIP (and ChIP-seq) has been used in
M. tuberculosis thus far for investigating transcription factor binding [12–14] and virulence factor binding [15, 16]. Others have
used advanced ChIP techniques such as ChIP-on-chip which
entails ChIP and hybridization to microarrays to study regulons
[14, 17]. Additionally, ChIP-seq has been used to complement
other techniques such as RNA sequencing to understand regulons
[18]. Here, we focus on the application of ChIP in M. tuberculosis
which can be used to study the binding patterns of a single and/or
multiple proteins to the genome such as transcription factor, transcription cofactors, histone modifications, and DNA repair
60
Heena Jagatia and Daire Cantillon