Abstract
In this study, GelGreen™ was investigated as a replacement for SYBR® Safe to stain DNA in cesium chloride (CsCl) density gradients for DNA-stable isotope probing (SIP) experiments. Using environmental DNA, the usage of GelGreen™ was optimized for sensitivity compared to SYBR® Safe, its optimal concentration, detection limit for environmental DNA and its application in environmental DNA-SIP assay. Results showed that GelGreen™ was more sensitive than SYBR® Safe, while the optimal dosage (15X concentration) needed was approximately one-third of SYBR® Safe, suggesting that its sensitivity was three times more superior than SYBR® Safe. At these optimal parameters, the detection limit of GelGreen™-stained environmental DNA was as low as 0.2 μg, but the usage of 0.5 μg environmental DNA was recommended to produce a more consistent DNA band. In addition, a modified needle extraction procedure was developed to withdraw DNA effectively by fractionating CsCl density gradients into four or five fractions. The successful application of GelGreen™ staining with 13C-labeled DNA from enriched activated sludge suggests that this stain was an excellent alternative of SYBR® Safe in CsCl density gradients for DNA-SIP assays.
Highlights
Stable isotope probing (SIP) is a technique that links microbial identity to metabolic function, and has proven to be a useful tool in microbial ecology
GelGreenTM was more sensitive than SYBR1 Safe as shown in Fig 3 where the fluorescence of DNA bands stained by GelGreenTM was stronger than those stained by SYBR1 Safe
SYBR1 Safe has been previously successfully used in environmental DNA staining in cesium chloride (CsCl) density gradients [9], the DNA bands stained by SYBR1 Safe in this study were too faint to be detected even if we strictly followed the recommendations of Martineau et al [9]
Summary
Stable isotope probing (SIP) is a technique that links microbial identity to metabolic function, and has proven to be a useful tool in microbial ecology. It works by incubating environmental samples with stable isotope labeled substrates (e.g. 13C) that when consumed are incorporated into the targeted biomarker molecules, such as phospholipid fatty acid (PLFA) [1], DNA [2] and RNA [3]. Application of PLFA-SIP is limited by the lack of knowledge of the profiles of the targeted molecules, especially in most uncultured microorganisms [4].
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