Target-probe hybridization

The probe-modified electrode is then immersed into a solution of target DNA to test its nucleotide sequence. When the sequence of target DNA exactly matches that of the probe DNA (based on the complementarity principle stating that A pairs with T and G with G), a hybrid (probe-target) duplex DNA is formed at the electrode surface. The following sections will focus on two most important steps in the detection of the DNA nucleotide sequence the formation of the DNA recognition layer and hybrid duplex DNA, and the transformation of the latter event into an electrical signal. 

Nucleic acid hybridization methods use oligonucleotide DNA probes with sequences complementary to a portion of the nucleic acid of the target bacterium38,60 and designed to hybridize with immobilized DNA or RNA on a membrane. After any unbound probe has been washed off, the hybridized probe can be detected.64 66... 

Lebaron, P. Catala, P. Fajon, C. Joux, F. Baudart, J. Bernard, L. A new sensitive, whole-cell hybridization technique for detection of bacteria involving a biotinylated oligonucleotide probe targeting rRNA and tyramide signal amplification. Appl. Environ. Microbiol. 1997, 63, 3274-3278. 

Arrays have been produced on filter supports, in microtiter plate wells and on glass slides coated and modified with one-, two- or 3-dimensional surface architectures as shown schematically in Figure 813 19. Glass offers a number of practical advantages, such as mechanical stability and low autofluorescence. Due to the non-porous character of glass chips, the volume of the hybridization solution can be kept to a minimum and probe-target interaction is not limited by diffusion into pores. However, three-... 

The acridinium ester (AE) in an AE-labeled cDNA probe hybridized to target DNA is less likely to be hydrolyzed than in the unhybridized conformation (Fig. 10) [9-11]. Single-base mismatches in the duplex adjacent to the site of AE attachment disrupt this protection, resulting in rapid AE hydrolysis [11]. Hydrolysis by a weak base renders AE permanently nonchemiluminescent. After hydrolysis, it is possible to use the remaining chemiluminescence as a direct measure of the amount of hybrid present. This selective degradation process is a highly specific chemical hydrolysis reaction, which is sensitive to the local environment of the acridinium ester. The matched duplex can be detected and quantified readily, whereas the mismatched duplex produces a minimal signal. 

For this hybridization strategy to work, the two probes (the capture and the labeled probe) should be from two adjacent portions on the genome, but without having complementary regions, thus avoiding binding of probes to each other. This hybridization requires target sample to bind to both the capture and the labeled probe and, as such, is more specific than direct hybridization on a membrane filter. 

Sonicated and denatured salmon sperm DNA (or other anionic maCTomolecules) may be used to reduce nonspecific probe interaction and electrostatic forces. The latter also may be reduced with dextran sulfate. High-stringency (low-sodium) hybridization ensures that complete complementarity will characterize the probe-target hybrid. 

Cellular autoradiography techniques using radioactive nucleic acid probes have several features in common with nucleic acid immunocytochemistry. The method is based on the hybridization of radioactive probes to cellular targets and the subsequent exposure of photographic emulsion, which, when developed, reveals blackened (exposed) silver grains close to the site of hybridiza-hon. Hence, cellular autoradiography techniques permit excellent specihcity and localizahon of the hybridized probe—to 1 qm when tritium is the label used in the autoradiography-based method (9). 

Nevertheless, it is clear from the work that the inclusion of betaine into the print buffer was an improvement over SSC or the MSS on several fronts. First, the SSC-betaine spotting was formd to increase hybridization efficiency as measured by a 2.5-fold higher hybridization signal intensity for probe-target hybrids relative to those probes spotted in SSC or MSS alone (Figure 4.34). 

In situ hybridization buffer is used for two purposes (1) probe denaturing and hybridization and (2) AP enzyme inactivation between two sequential AP-based BISH detections. As we previously reported (4), we learned that AP enzyme can be inactivated by incubating the tissue section with an in situ hybridization buffer containing formamide for 30 min at room temperature without losing probe-target hybridization. Thus, after the first AP-based BISH detection, an in situ hybridization buffer should be applied for inactivating prior to the second AP-based BISH detection. Since formamide can denature proteins, the slides must be washed well with a rinse solution before the second immunodetection step. 

PNA strand, resulting in an increase of the electrochemical signal (SWV peak current) as a result of probe-target hybridization. The PNA-functionalized conductive polymer sensor allowed for a detection limit of approximately 10 nM, and the feasibility for single-nucleotide mismatch detection was also demonstrated. 

The PCR procedure has an elegant simplicity. Two synthetic oligonucleotides are prepared, complementary to sequences on opposite strands of the target DNA at positions just beyond the ends of the segment to be amplified. The oligonucleotides serve as replication primers that can be extended by DNA polymerase. The 3 ends of the hybridized probes are oriented toward each other and positioned to prime DNA synthesis across the desired DNA segment (Fig. 9-16). (DNA polymerases... 

Similar techniques can be used to devise avidin—biotin assay systems for detection of nucleic acid hybridization. DNA probes labeled with biotin can be detected after they bind their complementary DNA target through the use of avidin-labeled complexes (Bugawanefrz/., 1990 Lloyd etal., 1990). Direct detection of hybridized probes can be accomplished, in a manner similar to that for LAB, by incubating with an avidin-enzyme conjugate followed by substrate development. BRAB-like and ABC-like assays also can be utilized to further enhance a DNA probe signal (Chapter 17, Section 2.3). 

Essentially, the CARD protocol is based on the deposition of haptenized tyramide molecules in the vicinity of hybridized probes catalyzed by horseradish peroxidase. The success of this technique depends on the integrity of target mRNA in sections and the ability of the probe to penetrate the sections and hybridize with mRNAs. Another requirement is an efficient reporter system capable of revealing low numbers of probe-mRNA hybrids per cell accompanied by low background staining. 

One of the most sensitive method for detecting probe-target hybrids involves an alkaline phosphatase-conjugated anti-DIG antibody and chemoluminescent alkaline phosphatase substrates (Roche, Molecular Diagnostics). Following hybridiza-... 

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