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Innovations in Noninvasive Instrumentation and Measurements
Published in Robert B. Northrop, Non-Invasive Instrumentation and Measurement in Medical Diagnosis, 2017
FISH is a molecular labeling technique which is used to detect the chromosomal location (and presence) of a specific genomic target. FISH was invented at the Lawrence Livermore National Laboratory in the mid-1980s by J.W. Gray and D. Pinkel. The FISH technique involves the selective binding of one or more probe molecules, each labeled with a separate, unique fluorescent dye (fluorochrome) to target single strands of DNA or RNA. The single-strand, labeled, DNA (or RNA) probe molecules are synthesized in lengths dependent on the application. Multiple complementary probes can be synthesized from all of the fragments from a single, long strand of DNA of a particular chromosome. These probes can label the total target chromosome. Alternately, chromophore-labeled probes can be made for a single gene, or can use a relatively short oligonucleotide, composed of as few as eight nucleotides. Short oligonucleotide probes can be used to find and label the complementary target sequences in any single-strand, chromosomal DNA or RNA.
Elucidation of Microbial Diversity in Wastewater Treatment System Through Molecular Tools: Molecular Tools, Techniques, and Applications in Wastewater Treatment
Published in Maulin P. Shah, Wastewater Treatment, 2022
Nikhil Nair, Sanchita Patwardhan, Nilesh S. Wagh, Jaya Lakkakula, Nomvano Mketo
There are several molecular methods including 16S rRNA polymerase chain reaction (PCR)-based amplification, denaturing gradient gel electrophoresis (DGGE), fluorescence in situ hybridization (FISH), DNA microarray, zeta-valent irons, etc., which are currently employed by many wastewater treatment plants (WWTPs) to study microbial diversity and microbial composition [4]. PCR is a powerful tool used in molecular biology to amplify a target sequence of interest for further quantitative studies. The 16S rRNA sequencing method is used to identify the organism from commercially available 16S rRNA sequence data with the help of 16S RNA obtained from targeted the microbial community. DGGE is a molecular fingerprinting technique that is employed to generate fingerprints with the help of denaturing agents like urea or formamide. The fingerprints obtained with DGGE help to study the phylogeny of microbial communities [4]. The FISH technique uses a fluorescently labeled complementary DNA/RNA probe that is altered such that it attaches to a particular region of targeted DNA/RNA. FISH helps to understand the chromosome location, morphology of the organism, or identification of the entire organism [5]. DNA microarray is an emerging technology used for the analysis of nucleic acid of microbes by considering rRNA genes as phylogenic markers [6]. The other less frequently used molecular tools for identification and quantification of microbial communities are ZVI, automated approach for ribosomal intergenic spacer analysis, the single-stranded conformation polymorphism (SSCP), random amplification of polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), terminal restriction fragment length polymorphism (T-RFLP), etc.
Diagnosing Microbiologically Influenced Corrosion
Published in Torben Lund Skovhus, Dennis Enning, Jason S. Lee, Microbiologically Influenced Corrosion in the Upstream Oil and Gas Industry, 2017
Jason S. Lee, Brenda J. Little
rRNA is essential for protein synthesis in all living organisms. In hybridization techniques, DNA or RNA is spotted onto a membrane and hybridized with labeled oligonucleotide probes (dot-blot hybridization). Whole cell hybridization or fluorescence in situ hybridization (FISH) can be used to detect and quantify particular microorganisms microscopically and to demonstrate their spatial relationships. Cells are fixed, incubated with an oligonucleotide probe labeled with a fluorochrome specific for 16S rRNA, and viewed with a fluorescence microscope.
Chromosomal damage in occupationally exposed health professionals assessed by two cytogenetic methods
Published in Archives of Environmental & Occupational Health, 2023
Dita Kadlcikova, Petra Musilova, Hana Hradska, Miluse Vozdova, Marketa Petrovova, Marek Svoboda, Jiri Rubes
The classical chromosomal aberration analysis (CA) of solid stained metaphase chromosomes of peripheral blood lymphocytes is a worldwide standardized method for human mutagenicity biomonitoring.4,8 Structural aberrations are recognized as anomalies in the conformation within a chromosome (ie, chromatid or chromosome break, ring chromosome) or a group of chromosomes (ie, chromatid or chromosome exchange, fragmentation, complex rearrangement). The asymmetric nature of the majority of structural aberrations detected by solid staining makes cells unstable during mitosis and their yield rapidly decreases with time after exposure.9,10 After its development in 1986, the fluorescence in situ hybridization technique (FISH) has fundamentally facilitated cytogenetic analysis by using painting probes. The FISH is able to detect unstable structural chromosomal aberrations (ie, dicentric chromosomes and acentric fragments) as well as stable ones (ie, translocations and insertions) which are transmitted to daughter cells during mitosis and tend to accumulate with repeated or chronic exposure.11–13
Biofilm formation by sulphate-reducing bacteria on different metals and their prospective role in titanium corrosion
Published in Environmental Technology, 2023
In the present study the strain of SRB was characterized and identified using standard SRB FISH probes. FISH probes are short sequences of single-stranded DNA, labelled with fluorescent markers, which are used to detect complementary DNA sequences of interest [19]. These probes hybridize, or bind, to the DNA and allow locating the DNA sequence. FISH is highly specific in spotting bacteria and their spatial variation in a biofilm community. Nucleotide probes with full-length 16S and/or 23S rRNA genes and labelled with fluorescent compounds were used in the present study to detect the SRB cells present in biofilm grown on glass and other metal substrates [13]. Figure S1 (see supplementary data) describes the SRB-specific probe-stained CLSM images, showing high specificity towards the bacterium Desulfovibrio vulgaris. The three different images recorded from the SRB biofilm illustrate the probe functionality and authenticate the purity of the SRB strain used for the study. In addition, the positive result of the desulfoviridin test confirmed the SRB strain to be the Desulfovibrio species. This test clearly differentiates the Desulfovibrio genus from other SRB types. The cell motility and curved rod-shaped cells, positive catalase activity furthermore confirm the strain as Desulfovibrio vulgaris.
Sulfate-reducing bacteria in anaerobic bioprocesses: basic properties of pure isolates, molecular quantification, and controlling strategies
Published in Environmental Technology Reviews, 2018
Ze-hua Liu, Hua Yin, Zhang Lin, Zhi Dang
Molecular microbiological techniques targeting 16S rDNA or functional genes were widely used for microbial quantification, which have provided greater fundamental understanding of bacteria in one specific system, bringing to light the importance of interested microbes. These include hybridization-based techniques as well as PCR-based techniques [79]. Among them, qPCR and fluorescence in situ hybridization (FISH) are two important methods. FISH is the most widely applied method through direct visualizing uncultured microbes, providing semi-quantitative information as well as detection of active microorganisms [80]. Disadvantages of FISH are time-consuming, limitation of ambiguous specificity, and high limit of detection [81]. Compared to FISH, advantages of qPCR are: (1) time-saving. The qPCR method only takes several hours from DNA extraction to a final result; (2) easy for operation. It can be performed without special skills or training; (3) more sensitive. Small quantities of DNA or old samples can be used; (4) high-throughput quantification and cost-effective [82,83]. Both qPCR and FISH have been applied in SRB studies [34,84–88]; when compared to FISH, qPCR is less applied but have much more application potential. Therefore, related information for SRBs based on the qPCR method was specially summarized.