Techniques

Techniques used in research on Anaerobic Methane Oxidation

FISH (Fluorescent in Situ Hybridization)

The FISH technique utilizes DNA probes that are specific to regions of individual DNA fragments. The probe attaches to the DNA of a bacteria, then a fluorescein stain is applied. This "paints" the DNA so that it is visible with the aid of a fluorescent microscope.

DGGE (Denaturing Gradient Gel Electrophoresis)

Denaturing gradient gels are used to detect non-RFLP polymorphisms. The small (200-700 bp) genomic restriction fragments are run on a low to high denaturant gradient acrylamide gel; initially the fragments move according to molecular weight, but as they progress into higher denaturing conditions, each (depending on its sequence composition) reaches a point where the DNA begins to melt. The partial melting severely retards the progress of the molecule in the gel, and a mobility shift is observed. It is the mobility shift which can differ for slightly different sequences (depending on the sequence, as little as a single bp change can cause a mobility shift).

T-RFLP (Restriction Fragment length polymorphism)

Restriction Fragment Length Polymorphism (RFLP) is a technique in which organisms may be differentiated by analysis of patterns derived from cleavage of their DNA. If two organisms differ in the distance between sites of cleavage of a particular restriction endonuclease, the length of the fragments produced will differ when the DNA is digested with a restriction enzyme. The similarity of the patterns generated can be used to differentiate species (and even strains) from one another.

PCR (Polymerase Chain Reaction)

The purpose of a PCR (Polymerase Chain Reaction) is to synthesize a huge number of copies of a gene. This amplification is required to have sufficient DNA fragments for cloning or for sequencing.
The PCR comprises of three major steps (denaturation, annealing and extension, repeated in up to 40 cycles. This reaction is performed on an automated cycler, called PCR machine, designed to heat and cool the tubes with the reaction mixture in programmed time intervals.

Q-PCR (Quantitative-Polymerase chain reaction)

Real–time or quantitative PCR (QPCR) allows quantification of starting amounts of DNA, cDNA, or RNA templates. QPCR is based on the detection of a fluorescent reporter molecule that increases as PCR product accumulates with each cycle of amplification. Fluorescent reporter molecules include dyes that bind double-stranded DNA (i.e. SYBR Green I) or sequence–specific probes (i.e. Molecular Beacons or TaqMan® Probes).

QPCR exceeds the limitations of traditional end–point PCR methods by allowing either absolute or relative quantification of PCR product at the end of each cycle. This ability has greatly enhanced several areas of research including gene expression analysis and genotyping assays.

Cloning and sequencing

DNA sequencing is the determination of the precise sequence of nucleotides in a sample of DNA. With a clone library a phylogenetic tree can be constructed in order to see resemblance with other organisms in a specific group or clade.

Lipid analysis

Incubating AOM capable sediments or enrichments with 13C labeled methane can show if there is incorporation of carbon from methane into the biomass. Another method focuses on the presence of specific lipids that only occur in anaerobic methanotrophs. Lipid biomarkers with extremely low d13C signatures indicative of anaerobic methanotrophy are found in the marine environment. Most of the studies on lipid biomarkers associated with AOM primarily focused on the presence and carbon isotopic compositions of a set of biomarkers derived from archaea: crocetane, pentamethylicosane, archaeol, hydroxyarchaeol, and glycerol tetraethers. In contrast, existing data on lipid biomarkers proposed to derive from the SRB partners involved in AOM suggest that SRB biomarkers are also highly diverse and include straight chain and methyl-branched fatty acids and alcohols as well as mono- and dialkylglycerolethers. Other important information has been acquired by lipid analysis of Black Sea microbial mats that provided evidence that also hopanoid-producing organisms may be involved in AOM activity. The inferred presence of these compounds to a permanently anoxic environment is significant because such lipids are not known to occur in strictly anaerobic bacteria.

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