Abstract
Understanding the genetic safeguarding mechanism of Mycobacterium tuberculosis (Mtb) may help us to explain i), how Mtb survive the genetic assaults elicited by both reactive oxygen species (ROS) and reactive nitrogen species (RNS) produced by host macrophages and ii), why some strains of Mtb, e.g., Mtb strains from East Asian lineage and Beijing sublineage, exhibit high mutation rate and are more likely to acquire drug resistant mutations (e.g., rifampicin-resistance mutation) during infection. Mutation frequency analysis is a basic methods to study the genetic safeguarding mechanism. Moreover, to study the molecular mechanism of mutation, it is necessary to analyse the mutation spectrum (For example, oxidized cytosine may induce CG to TA mutation.). This protocol describes a method to determine the mutation frequency and understand the mutation spectrum in both Mycobacterium smegmatis (Msm) and Mtb.
Keywords: Mycobacterium, Mutation frequency, DNA damage
Materials and Reagents
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Acknowledgments
This protocol was adapted from a previously published paper Lyu et al. (2013). This work was supported by grants from the National Natural Science Foundation of China (No. 30970077, 31121001, 31300126), the Research Unit Fund of Li Ka Shing Institute of Health Sciences (No. 7103506), the Hong Kong Health and Medical Research Fund (No. 12110622), the China Postdoctoral Science Foundation (No. 20110490754), the SIBS Postdoctoral Research Fund (No. 2011KIP509).
References
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