Membrane hybridization

EJ-Haiwagi, M. M. (1993). Optimal design of membrane hybrid systems for waste reduction. Sep. Sci. TechnoL, 28(1-3), 283-307. 

Muralikrishnan, G., Crabtree, E., and El-Halwagi, M. M. (1996). Design of Membrane Hybrid Systems for Pollution Prevention." AIChE Spring Meeting, New Orleans, LA. 

Clearly, bDNA is more sensitive than LH, HC, and in-house membrane hybridization assays, but its relatively high LOQ is not optimal for detecting lower titers associated with active liver disease. There have been no differences in clinical specificity among the HBV DNA quantitation methods reported. 

Figure 11 Chemiluminescent detection for membrane hybridization of unmodified DNA target by derivatization reaction with TMPG. Procedure [15] A portion of the DNA solution is spotted on a nylon membrane. The target DNA is hybridized to its cDNA probe having a -(G)15TT(G)15TT at its 3 terminus in a hybridization buffer (pH 7.0) at 42°C for 2 h. After washing, the membrane is moistened with sodium phosphate solution (pH 10) for a few seconds, and then immersed in 0.2 M TMPG dissolved with dimethyl sulfoxide for 0.5 min at ambient temperature. The moist membrane is then dipped in dimethyl-formamide for a few seconds, and the luminescence is detected for 0.5 min.

In the tissue sections, a hybridization is performed with a highly specific probe in order to detect the target (either amplified by indirect in situ RT-PCR or unamplified). For the preparation of nonradioactive digoxigenin-labeled probes, one should refer to the guidelines of the company manuals Genius M System User s guide for membrane hybridization and Nonradioactive in situ hybridization application manual (2nd ed., Boehringer Mannheim). 

Koizumi, Y. Kelly, J.J. Nakagawa, T. Urakawa, H. El-Fantroussi, S. Al-Muzaini, S. Fukui, M. Urushigawa, Y. Stahl, D.A. Parallel characterization of anaerobic toluene- and ethylhenzene-degrading microhial consortia hy PCR-denaturing gradient gel electrophoresis, RNA-DNA membrane hybridization, and DNA microarray technology. Appl. Environ. Microbiol. 2002, 68, 3215-3225. 

J.C. Davis, R.J. Valus, R. Eshraghi and A.E. Velikoff, Facilitated Transport Membrane Hybrid Systems for Olefin Purification, Sep. Sci. Technol. 28, 463 (1993). 

Pressly, T.G. Ng, K.M. A break-even analysis of distillation-membrane hybrids. AIChE. 1998,44,93-105. 

Gottschlich, D.E. and Roberts, D.L. (1990) Energy Minimization of Separation Processes using Conventional/Membrane Hybrid Systems, Department of Energy Report No. DE-AC-07-76ID01570. 

Davis JC, Valus RJ, Eshraghi R, and Vilikoff AE. Facilitated transport membrane hybrid systems for olefin purification. Separation Science and Technology 1993 28(1-3) 463-476. 

The remaining cells are used to isolate genomic DNA for filter membrane hybridization to determine EBV genome copy numbers per cell (see Appendices 2 and 4). 

Five steps can be distinguished in membrane hybridization (i) immobilization of target nucleic acid (ii) prehybridization to saturate the remaining binding sites which would otherwise adsorb probe non-specifically (iii) hybridization in low stringent conditions to adsorb probe as efficiently as possible (iv) posthybridization washes to define the stringency of the hybridization and thus the specificity of the reaction (v) the detection step (Fig. 8.2). In addition, the hybridized probes can sometimes be stripped from the blots to expose the targets to other probes. 

Some polymers can be used as accelerators of nucleic acid hybridization. They sometimes produce background staining and are only recommended when the hybridization rate is low (small amounts of probe or target). Dextran sulfate is often used as an accelerator but polyethylene glycol (PEG 6000) and sodium polyacrylate have been shown to be valuable alternatives. These polymers probably act by exclusion of probe molecules from the volume occupied by the hydrated polymer which results in an effective increase in the probe concentration (Wetmur, 1975). The optimal probe concentration in membrane hybridization is considered to be about 10 cpm of probe/ml (1-10 ng/ml), but Amasino (1986) found a 2- to 10-fold reduction in the probe concentration to be optimal when 10% PEG was included (note that these effects may depend on the specific activity of the probe). These polymers do not act in a similar way and have their own characteristics. 

A wide variety of hybridization conditions can be expected for this heterogeneous group of solid phases. The hybridization time depends on the nature of the solid phase and complexity of the strand in excess and may range from that comparable to membrane hybridization (Section 8.2 e.g., microtiter plates and polynucleotide probes), to a few minutes (small particulate solid phase will display a hybridization rate which approaches that of solution hybridization Table 3.16). 

Two methods were used to quantitate residual host cell (HEK 293) DNA in AdSFGF-4 preparations. The first, utilizing membrane hybridization of an oligonucleotide probe followed by chemiluminescence detection, was based on a previously de-... 

A EXPERIMENTAL FIGURE 9-16 Membrane-hybridization assay detects nucleic acids complementary to an oligonucleotide probe. This assay can be used to detect both DNA and RNA, and the radiolabeled complementary probe can be either DNA or RNA. 

Clearly, Identification of specific clones by the membrane-hybridization technique depends on the availability of complementary radiolabeled probes. For an oligonucleotide to be useful as a probe, it must be long enough for Its sequence to occur uniquely In the clone of interest and not In any other clones. For most purposes, this condition Is satisfied by oligonucleotides containing about 20 nucleotides. This Is be-... 

The PCR depends on the ability to alternately denature (melt) double-stranded DNA molecules and renature (anneal) complementary single strands in a controlled fashion. As in the membrane-hybridization assay described earlier, the presence of noncomplementary strands in a mixture has little effect on the base pairing of complementary single DNA strands or complementary regions of strands. The second requirement for PCR is the ability to synthesize oligonucleotides at least 18-20 nucleotides long with a defined sequence. Such synthetic nucleotides can be readily produced with automated instruments based on the standard reaction scheme shown in Figure 9-18. 

Many industrially important liquid systems are difficult or impossible to separate by simple continuous distillation because the phase behavior contains an azeotrope, a tangent pinch, or an overall low relative volatility. One solution is to combine distillation with one or more complementary separation technologies to form a hybrid. An example of such a combination is the dehydration of ethanol using a distillation-membrane hybrid, as shown in Figure 6.30. 

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