Optimized DNA Preparation from Mycobacteria

Abstract

Extraction of genomic DNA from mycobacteria requires special consideration because (i) many mycobacterial species exhibit extremely slow growth, and thus produce only small amounts of starting material, and (ii) a robust and waxy cell wall renders mycobacteria difficult to lyse. Hence, mycobacterial DNA extraction often results in low DNA yields of unsuitable quality. Published protocols for mycobacterial DNA preparations and commercially available extraction kits are mainly designed for the isolation of small amounts of genomic material suitable for polymerase chain reaction (PCR)-based applications like species identification. However, such DNA quantities and qualities are usually not sufficient for contemporary genomic analyses such as whole genome sequence analysis, single nucleotide polymorphism (SNP) detection, or DNA microarrays, or for investigations of bacterial evolution, virulence, or epidemiology on a world-wide population level. Moreover, most protocols that achieve a high standard in DNA recovery typically employ large reaction volumes and thus require milliliter-scale plasticware and centrifugal equipment as well as large amounts of chemicals, all of which are costly both in purchase and disposal. The DNA extraction method described here was established to address the challenges that result from the slow growth and distinct cell wall composition of mycobacteria, and to greatly enhance both yield and purity of mycobacterial DNA preparations in a small extraction volume. Designed to be performed using 1.5-mL reaction tubes and the corresponding equipment, the method is economical and practical, and reliably yields large amounts of pure genomic DNA--increases of at least 10-fold as compared to earlier protocols.

Genomic characterization of Nontuberculous Mycobacteria

“…Purified genomic DNA was obtained using a specific protocol for mycobacteria including enzymatic digestion, mechanical disruption of the cell wall and extraction with phenol/chloroform/isoamyl alcohol 25:24:158. To confirm the strains identity hsp65, rpoB and 16S rRNA genes were partially sequenced by the Sanger method.…”

Retrospective observational study of diagnostic accuracy of the Xpert® MTB/RIF assay on fiberoptic bronchoscopy sampling for early diagnosis of smear-negative or sputum-scarce patients with suspected tuberculosis

“…This assay requires less than 2 hours, and its key advantage over other PCR methods is that it is a fully-automated process, designed to run on the GeneXpert Dx system (Cepheid). This system incorporates DNA extraction, often considered the critical step [15], along with real-time PCR amplification and detection in a single hands-free process, thus acting as a real “lab-on-chip” device.…”

Development of a Temperature-Switch PCR-Based SNP Typing Method for Mycobacterium ulcerans

“…Ten of these isolates, used for the setup of SNP typing assays, had been typed previously by real-time PCR as SNP haplotypes 1–10 (Agy99 (HT1), NM98/03 (HT2), NM83/03 (HT3), NM100/03 (HT4), NM27/02 (HT5), NM18/02 (HT6), NM74/03 (HT7), NM32/02 (HT8), NM28/02 (HT9), and NM78/03 (HT10)) [13]. Genomic M. ulcerans DNA was isolated by cell wall disruption and phenol-chloroform extraction as described earlier [14]. DNA was quantified by using Qubit Fluorometer (dsDNA HS Assay Kit, Invitrogen).…”