Competitive hybridization

The design of hybridization formats that enable important changes (fluorescence intensity increase or wavelength shifts) in the presence of the targets is the focus of intense research because their use can simplify the analyses in vitro and provide the possibility of applications in living organisms. The most frequently used involve binary probes (35), competitive hybridization probes (18), linear probes with only one F (2, 5, 16, 29, 31), and molecular beacons (MBs) (39-41), (Fig. 3). 

Figure 3 Schematic representation of the main hybridization formats, (a) Binary probes, (b) Competitive hybridization probes, (c) Linear probe with one F. (d) Moiecuiar Beacon, (e) Aptamer.

By using a multistep procedure, DNA molecules have been covalently attached to SWNTs. First, the purified SWNTs were oxidized to form carboxylic acid groups at the ends and sidewalls, followed by reaction with thionyl chloride and ethylenediamine to produce amine-terminated sites. The amines were then reacted with the heterobifunctional cross-linker succinimi-dyl 4-(iV-maleimidomethyl)cyclohexane-l-carboxylate (SMCC), leaving the surface terminated with maleimide groups. Finally, thiol-terminated DNA reacted with these groups to produce DNA-modified SWNTs [161]. AAHien DNA is covalently attached to SWNTs, a better stability, accessibility and selectivity are expected during competitive hybridization. 

As mentioned previously, one of the main differences between one- and two-channel arrays is that the latter are based on the competitive hybridization of two RNA samples with the probes on the array. In practice, this implies that two-color arrays are intended to quantify how much each gene is expressed in one sample relatively to the other. 

Figure 4.1 Voltammetric detection of DNA oligonucleotides. After immobilization of the ssDNA probe on gold (black), the electrode surface was additionally covered with mercaptobutanol (gray) to prevent unspecific binding. Competitive hybridization of MB-labeled reporter and nonlabeled target ssDNA. The amount of hybridized reporter DNA is gauged by differential pulse voltammetry. [From Panke (2007).]...

Panke et al. [69] performed a different approach related to a competitive binding protocol for the determination of DNA single base mismatches by using methylene blue in combination with differential pulse voltammetry technique. Duwensee et al. [70] reported a strategy for sequence-specific DNA detection by means of a competitive hybridization assay with osmium tetroxide-labeled signaling probes. 

H. Duwensee, M. Jacobsen, and G. U. Flechsig, Electrochemical competitive hybridization assay for DNA detection using osmium tetroxide-labelled signalling strands, Ana/yst 134[5], 899-903 [2009]. 

In studies with normal cells, unlabeled HnRNA is hybridized to DNA on filters, which are then washed and reexposed to radioactively labeled cytoplasmic mRNA. If identical sequences are present in the two fractions then hybridization of the labeled species will be inhibited since the relevant DNA sites will have already been filled. This technique of competitive hybridization has shown that some sequences in HnRNA are also present in mRNA (e.g., Soeiro and Darnell, 1970). However, the conditions used in most of these experiments have only allowed the hybridization of RNA to the highly reiterated fraction of DNA, in which certain DNA sequences are present in thousands of similar, nonidentical copies (Britten and Kohne, 1968). It is therefore possible that the competing RNA sequences are also similar, but not necessarily identical, and in any case may represent only a small fraction of the total sequences. This difficulty has to some extent been overcome by using more stringent conditions which allow hybrids to form between RNA and the less frequent DNA sequences (Scherrer et ah, 1970 Darnell et ah, 1970). Again, evidence has been obtained for a precursor-product relationship between HnRNA and cytoplasmic mRNA. 

The most likely interpretation of these results is that tlie viral DNA is first transcribed into a large precursor RNA molecule which is specifically cleaved in the nucleus before or during its transport into the cytoplasm. Competitive hybridization studies have shown that sequences present in virus-specific mRNA are indeed present in the large nuclear RNA s. In some cases there is evidence for sequences in the nuclear RNA s which are not present in the cytoplasmic RNA, suggesting that the processing is not necessarily completely conservative (Wagner and Roizman, 1969 McGuire et al., 1972). 

Combination of europium- and terbium-labeled oligonucleotide probes with complementary probes labeled with quenchers has been used in competitive hybridization assay as to screen celiac disease-related alleles [23] and cystic fibrosis risk alleles [42]. Doubly labeled beacon-type probes containing europium chelate as donor and either Alexa 647 or Alexa Fluor 700 as acceptors has been utilized by measuring both the wavelength and decay change upon hybridization [43]. 

Several methods are available to monitor transcription levels of tens of thousands of genes rapidly and simultaneously. The quantification of RNA species by sequence-specific annealing (hybridization) to complementary DNA probes arrayed on a substrate (microarray) was developed by Schena etal. (1995). Sample RNA was submitted to reverse transcription and fluorescent labeling. Thereby, a quantitative parameter was produced that could be measured as a localized signal after hybridization to the arrayed sensors. Comparison by competitive hybridization of two RNA samples labeled with two different dyes (Cy3 and Cy5) resulted in expression ratios of the two sources (e.g., of tumor and nontumor tissue). Whereas sensors were originally taken from libraries of DNA clones (cDNA), present-day microar-... 

The DNA of any given AAV serotype will hybridize approximately 33% with the DNA of anj of the other serotypes as judged by DNA RNA hybridization assays using nitrocellulose filters (Rose et al, 1968). Further studies by Koczot (1970) using competitive hybridization have shown that the related sequences are the same for all four serotypes. More recent competitive DNA DNA hybridization studies have indicated that the homology among the different serotypes is closer to 50% (Koczot, personal communication). 

Morrison, L. E. Haider, T. C. Stols, L. M. Solution-phase detection of polynucleotides using interacting fluorescent labels and competitive hybridization. Anal. Biochem. 1989,183, 231-244. 

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