Specificity

Superior specificity has been achieved with the Invader technology by combining sequence identification with enzyme structure recognition. The key to the assay's specificity is the strict requirement for an invasive structure. If the sequence at the cleavage site is not complementary to the intended target, an invasive structure does not form, and the 5'-flap is not released for detection in the secondary reaction. For mutation detection applications, such as cystic fibrosis transmembrane conductance regulator (CFTR) gene screening, this discrimination capability provides high accuracy, as demonstrated by its ability to classify the three possible CFTR AF508 genotypes and discriminate between the AI507 and AF508 mutations (Fig. 3). In a multisite study to determine the accuracy of genotyping five polymorphisms associated with venous thrombosis, the Invader assays correctly genotyped 99.9% of samples compared with 98.5% correct genotypes called by PCR-based methods.[7] Such ability to discriminate single-base changes also makes the Invader platform ideally suited for genotyping highly homologous strains of infectious agents.

The Invader platform is capable of specifically geno-typing highly polymorphic regions such as the cyto-chrome P450 gene family, which contain multiple pseudogenes, gene deletions, and gene duplications. To prevent false positive results or inflated wild-type signals caused by association of Invader oligos to the pseudo-genes, the region of interest can be amplified using PCR primers specific to the region of interest. The resulting amplicons can be used as templates for Invader assay-

based detection. Using this approach, results from 11 Invader CYP2D6 assays provided a clear genotype for 100% of the 171 anonymous donor samples that had a visible PCR product on an agarose gel.[5]

The quantitative aspect of the Invader platform allows for the determination of chromosome and gene copy number directly from genomic DNA. This analysis may be accomplished by comparing the specific signal generated from the gene or chromosome of interest with that of a reference gene such as a-actin, which is not known to be polymorphic for either duplication or deletion.[5] Careful design of the Invader oligos to regions with minimal homology to other regions within the genome enables accurate quantitation of the desired target sequence.

Invader RNA assays have the same ability as the DNA assays to discriminate single-base changes. By positioning the cleavage site of the primary probe at a nonconserved site within the mRNA target, Invader assays are able to differentiate highly homologous RNA sequences. Cross-reactivity is undetectable in the presence of 10,000-fold excess of highly related mRNA.[4] In addition, assays with cleavage sites located at particular exons or splice junctions can distinguish alternatively spliced mRNA variants.[6]

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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