False positives hybridization

To be sure, molecular genetics suffers from limitations. Many of its methods cannot be used without the availability of at least one known amino acid sequence. Thus, peptide isolation remains a critical need. Oligonucleotide probes can be too short, leading to useless or "false positive" hybridizations. Also, an appropriate bioassay is required to assess the authenticity of any product of molecular biology. However, the use of molecular genetic techniques will cause an explosion of information on insect neuropeptides and their sequences within the very near future. 

A major challenge in the use of two-hybrid systems is the elimination of false positives. These clones result from activation of reporter... 

As a result of this protocol, four indicators were dropped because in each case, they did not pass the first consistency test, that is, failed to discriminate adequately at all levels of the scale. Next, Tyrka et al. (1995) calculated the taxon base rate for each indicator using a hybrid of MAXCOV and Latent Class Analysis estimation procedures (for details see Golden, 1982) and adjusted the estimate for the true- and false-positive rates computed earlier. The average taxon base rate was. 49. The authors did not report a variability statistic, but a simple computation shows that SD of base rate estimates was. 04. 

A simple, fast and specific color test for urea nitrate was reported recently by Almog et al. It is based on the reaction between urea nitrate and ethanolic solution ofp-dimethylaminocinnamaldehyde (p-DMAC) (9) under neutral conditions [91]. A red pigment is formed within 1 min from contact. Its structure has also been determined by the same group, by X-ray crystallography [92]. It appears to be a resonance hybrid between a protonated Schiffbase (10) and a quinoid system (10a) (Eq. (14)). The limit of detection on filter paper is 0.1 mg/cm. Urea itself, which is the starting material for urea nitrate, does not react with p-DMAC under the same conditions. Other potential sources of false-positive response such as common fertilizers, medications containing the urea moiety and various amines, do not produce the red pigment with p-DMAC. 

In order to reduce the effects of cross-hybridization that lead to false positives, researchers at Affymetrix created a series gene-specific but closely... 

We compared the assay results for 80 common chemicals from both the Nishihara et al. and NCTR data sets inconsistent assay results were observed for 12 chemicals. Specifically, of 30 active chemicals in the Nishihara et al. data set, one chemical was found inactive in the NCTR data set of 50 inactive chemicals in the Nishihara et al. data set, 11 chemicals were found active in the NCTR data set. These observations show that even using the experimental data from the ER binding assay (the NCTR data set) to predict the experimental results from the yeast two-hybrid assay (the Nishihara et al. data set), there may be about a 15% (12/80) discrepancy, or 3.3% (1/30) false negative rate and 22% (11/50) false positive rate. Care should be taken in interpreting the QSAR validation results using this data set (Hong et al., 2002). 

Because of the sequence complexity of genomic DNA, isolation of false positive clones by blot hybridization is a significant problem when the stringency of the washing conditions is very low (wash temperature <42°C). 

Serebriiskii IG, Golemis EA. Two-hybrid system and false positives. Approaches to detection and elimination. Methods Mol. Biol. 2001 177 123-134. 

The first step in constructing a cell signaling network model is to generate an in silica interaction network. Signaling components of interest are identified, and data on binary interactions are extracted from the experimental literature. Often times, the creation of an entire interaction map for a large network is beyond the scope of one laboratory therefore, public databases have been created in which newly discovered proteins and/or protein interactions are deposited (57-63). However, often data are included from studies that cover a broad range of protein interactions, such as proteomic studies or yeast two-hybrid screens, and frequently contain many potential false positives or negatives. Thus, it becomes necessary to critically examine each reference to verify the interaction data reported, as this in silica network is the basis of further study. 

The dideoxy fingerprinting (ddF) is described by Sarkar et al. (92) to be a hybrid between dideoxy sequencing and SSCP. The method is rapid, large and small regions can be amplified and screened. In the initial publication, Sarkar et al. detected 84 out of 84 known mutations. The frequency of false positivity has been described to be low—in the order of 5% (92,97). The ddF technique has been explored on 73 primary breast cancers (97). Sarkar et al. claimed that this technique detected 100% of the gene mutations, but compared with SSCP, ddF technique requires 50% more effort (97). 

It is extremely important to control the quality of the two DNase digestions to ensure that the RNA template is free of residual EMCV plasmid DNA. Presence of EMCV DNA would result in false-positive results or background Amp-RT signal. To check for absence of EMCV DNA, the RNA template is subjected to PCR amplification and probing by Southern blot hybridization to 32P-labeled EmcPl probe. 

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. 

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