Nonspecific hybridization

The molecular sensitivities of the first and second generations of the bDNA assays were limited by nonspecific hybridization between the amplification probes and other nucleic acids. Short regions of hybridization between any of the probes constituting the amplification system, (preamplifier, amplifier, and labeled probe) and any nontarget nucleic acid sequence leads to amplification of the background signal. Capture probes, capture extenders, and sample nucleic acid are all sources of this background hybridization (Collins et al 1997). 

Nonspecific hybridization Does a noncomplementary probe of identical size and G-C composition hybridize Does a nuclease-treated specimen show hybridization Does protease treatment decrease hybridization ... 

DNA that was covalently bound to a polymer. The hybridized P-1 and P-2 DNA chains functioned as hydrophilic linkers in addition, they increased the negative electric charge density on the surface. For these reasons, our DNA-conjugated polymer-coated substrate showed better prevention of nonspecific hybridization of SNP sequences and was selective exclusively for fully matched DNA. 

The use of primary tissue preparations and plasmid probes may lead to nonspecific hybridization (Howell and Kaplan, 1987). Xenogeneic tissues are frequently cultivated in immunodeficient animals and may contain bacterial DNA (episomes, plasmids) and lead to erroneous conclusions. In addition to athymic mouse xenografts, bacterial contamination may occur in tissues from other sources such as patients immunocompromised by disease or chemotherapy (opportunistic infections) or even tissues from healthy individuals from organs rich in bacterial flora (e.g., the gut). Spurious hybridization is easily identified with vector probes and can be avoided using probes generated... 

Suppression hybridization is used when repeated DNA is present in both target and probe sequences. Since these repeat sequences are present in a relatively much higher concentration and since they have a lower complexity, they would give a much stronger signal than less abundant sequences. These nonspecific hybridization signals can be suppressed by hybridizing first with unlabeled repetitive DNA probes. Such probes are now commercially available for different species (e.g., from BRL). 

Although this amplification technique is very sensitive and has tremendous application potential, it is not without problems. The powerful amplification procedure may yield false-positive results when samples are contaminated by nucleic acid left over from previously amplified DNA. Other problems include primer artifact formation and nonspecific hybridization of primers to DNA samples. Several modifications to the original PCR technology have been made over the years to improve the sensitivity and application potential for PCR, including the use of multiple sets of amplification primers, multiplex PCR, PCR amplification of RNA by converting targeted RNA with reverse transcriptase to complementary DNA templates (which are then suitable for DNA amplification by traditional PCR techniques), and real-time quantitative PCR. 

Hybridizations can either occur at 65°C or 42°C (if 50% formamide is included). General and species specific blocking elements should be included in the hybridization. For human RNA-derived probes in a 25 pL hybridization, use of 10 pL of 1 mg/mL CoT-1 DNA helps to block repetitive human DNA, 1 pL of 4 mg/pL yeast tRNA acts as a nonspecific hybridization blocker and 1 pL of 8 mg/mL poly dA blocks the oligo dT. The CoT-1 DNA and the tRNA can be added to the probe just prior to purification. 

The oldest DNA technique in food analysis is based on labeled probes, which bind directly to DNA if complementary regions exist. This method, called Southern hybridization, is very time consuming. Further disadvantages are insufficient sensitivity for many questions, as well as the problem of nonspecific hybridization signals for closely related animal species (Behrens and Unthan, 1999). 

Regarding the interactions between bulk- and surface-phase kinetics mentioned above, Eq. 21 needs to be coupled with appropriate boundary conditions depicting specific and nonspecific hybridization, as applied to all reacting surfaces. In the present formulation, incorporation of these boundary conditions is effected by formulating the source term / , in Eq. 21 such that / , = 0 for the bulk phase. However, for the reactive bottom wall (where the capturing probes are attached). 

The addition of sheared, denatured heterologous DNA to the hybridization buffer has been found to reduce nonspecific hybridization, and it has therefore been included as a standard component. However, with probes showing a low level of crosshybridization to other sequences in genomic DNA it is possible to omit this component. This can result in increased specific signal. 

With Tda = 0.03 ms, we obtain 7 da = 4.2 nm. This is close to 4.1 nm obtained for the trivial case, i.e., the case where we assumed the presence of a nonspecific hybrid in the mixture instead of an aggregate with two acceptors. 

The labeled cDNAs are now exposed and allowed to hybridize with the probe DNA. This hybridization is similar to Southern blotting method. After that slide is washed properly, which removes nonspecific hybridization, it is read in a laser scanner that can dififerentiate between Cy3- and Cy5-signals, collecting fluorescence intensities to produce a separate 16-bit TIFF image for each channel (Venter and Adams 2001 Young 2000). 

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