A liquid chromatography - mass spectrometry method to measure ¹³C-isotope enrichment for DNA stable-isotope probing

Canadian Journal of Microbiology
Julie AuclairRichard Villemur

Abstract

DNA stable-isotope probing (DNA-SIP) is a cultivation-independent technique that makes it possible to associate metabolic function and taxonomic identity in a wide range of terrestrial and aquatic environments. In DNA-SIP, DNA is labeled via the assimilation of a labeled growth substrate that is subsequently used to identify microorganisms involved in assimilation of the substrate. However, the labeling time has to be sufficient to obtain labeled DNA but not so long such that cross-feeding of ¹³C-labeled metabolites from the primary consumers to nontarget species can occur. Confirmation that the DNA is isotopically labeled in DNA-SIP assays can be achieved using an isotope ratio mass spectrometer. In this study, we describe the development of a method using liquid chromatography (HPLC) coupled to a quadrupole mass spectrometer (QMS) to measure the ¹³C enrichment of thymine incorporated into DNA in Escherichia coli cultures fed with [¹³C]acetate. The method involved the hydrolysis of DNA extracted from the cultures that released the nucleotides, followed by the separation of the thymine by HPLC on a reverse-phase C₈ column in isocratic elution mode and the detection and quantification of ¹³C-labeled thymine by QMS. To mimic a DN...Continue Reading

References

Aug 25, 1995·Nucleic Acids Research·D W Hoffman, J A Holland
Nov 1, 1995·Analytical Biochemistry·M B Goshe, V E Anderson
Feb 25, 2000·Nature·S RadajewskiJ C Murrell
Mar 2, 2002·Applied and Environmental Microbiology·Samantha A MorrisJ Colin Murrell
Oct 31, 2002·Applied and Environmental Microbiology·Mike ManefieldMark J Bailey
Jun 1, 1962·Journal of Molecular Biology·C L SCHILDKRAUTP DOTY
Oct 31, 2006·Microbial Ecology·Josh D NeufeldJ Colin Murrell
Feb 15, 2007·Environmental Microbiology·Feth El Zahar HaicharOdile Berge
May 1, 2007·Applied and Environmental Microbiology·Helen W Kreuzer-Martin, Kristin H Jarman
Sep 14, 2007·Mass Spectrometry Reviews·Jean-Philippe GodinGérard Hopfgartner
Aug 30, 2008·The ISME Journal·Feth el Zahar HaicharWafa Achouak

❮ Previous
Next ❯

Citations

May 7, 2014·Rapid Communications in Mass Spectrometry : RCM·Tanja C W Moerdijk-PoortvlietHenricus T S Boschker

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.

© 2022 Meta ULC. All rights reserved