Hybridization capture

Amagliani, G., Omiccioli, E., del Campo, A., Bruce, I. ]., Brandi, G., and Magnani, M. (2006). Development of a magnetic capture hybridization-PCR assay for Listeria monocytogenes direct detection in milk samples. /. Appl. Microbiol. 100, 375-383. 

Identification, magnetic capture hybridization, cyanobacteria, photosynthetic bacteria, hydrogen production, double-phase reactor, oxygen effect... 

S rDNA targeted identification of cyanobacteria using magnetic capture hybridization... 

Magnetic capture hybridization has attracted considerable attention owing to the ease with which specific target DNA may be isolated from a heterogenous sample. This simple method furthermore permits rapid concentration of target DNA from a bulk sample. It would therefore allow sensitive detection of target DNA from environmental samples, blood or other clinical samples. 

Takeyama H, Tsuzuki H, Chow S, Nakayama H, Matsunaga T. (2000). Discrimination between Atlantic and Pacific subspiecies of the northembluefin tuna (Thunnus thynnus) by magnetic capture hybridization using bacterial magnetic particles. Mar. Biotechnol. 2,309. 

Purified nucleic acids are poor immunogens but can serve as high molecular weight haptens. Synthetic RNA DNA hybrids, such as poly(A) poly(dT), can serve as immunogens (Stollar and Rashtchian, 1987) and the antiserum obtained can be absorbed with ds RNA or denatured DNA. The remaining antibodies may specifically capture hybrids from solution or in competitive ELISA test. 

Sandwich hybridization, using affinity-based hybrid collection, is based on two nonoverlapping nucleic acid probes (one is labeled, the other can be collected by the affinity matrix) (Syvanen et al., 1986 Jalava et al., 1990). The principles are shown in Fig. 8.3. Target nucleic acid thus mediates binding of labeled probe to the matrix. The detectability is about 10 molecules with a linear range to at least 10 molecules with radioisotopes as labels. In contrast to capture hybridization assays, the immobilization of the complex is at 22-37°C (leaching is then usually less important). 

The basis of contemporary microarray technology is printing known oligonucleotide sequences in predefined positions onto the array surface, which then capture the labeled complementary strands from the sample, following the complementarity rules (duplex forming and hybridization). After hybridization, the original amount of mRNA molecules can be estimated from the signal intensity of the captured/hybridized molecules. In the case of the microscope slide-based microarrays (also referred to as cDNA microarray), presynthesized complementary DNA molecules are attached to a solid surface [40]. 

Magnetic capture hybridization offers several advantages among them are speed and strong binding between biotin and streptavidin. However, the high cost of magnetic beads and low recovery of the RNA are often the major draw-... 

Fig. 12 Description of the magnetic capture hybridization method. Hybridization is first performed between RNA and biotin-labeled probes. The hybridized RNA is separated from the unhybridized RNA using streptavidin-coated magnetic beads...

For example, a solid phase coated with avidin or streptavidin can be used to capture hybrids containing a biotinylated primer. Subsequent detection reactions involve the label that is attached to the probe (Figure 1). Solid phases also can be coated with monoclonal or polyclonal antibodies. Cost and availability are considerations that have prompted many researchers to use avidin. 

Within the last decade applications of new cidture-independent molecular tools based on PCR analysis of soil-extracted nucleic acids have provided unique insights into the conqiosition, richness and structure of microbial communities (58-61). Quantitative PCR methods will be used to estimate the abundance in soils of genes that encode atrazine-degradation enzymes, and by quantifying mRNA, their expression. Magnetic capture hybridization (MCH) followed by nested PCR could predict die potential of a soil to mineralize atrazine (62). In diis study, atzA gene copy number was quantified and found to be correlated... 

Bach, H.-J. et al.. Magnetic capture-hybridization method for purification and probing of mRNA for neutral protease ol Bacillus cereus. Journal of Microbiological Methods, 1999 37(2) 187-192. 

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