Sensitivity

Generically, analytical sensitivity is defined as the proportion of positive test results, when a detectable mutation is present. Analytical sensitivity is equivalent to the analytical detection rate. Between 1996 and 2001, when RLBs were widely used, Palomaki et al.[2] found an analytical sensitivity of 97.9%,[2] in U.S. CF molecular testing. They added that analytical sensitivity was consistent over the 6 years.

Two genetic tests, one for the hemoglobinopathies and p-thalassemia that would have broad (95% clinical sensitivity) worldwide coverage, and the other for a series of relatively common mutations in the human CFTR, were the first application of RDBs. The clinical sensitivity of the 25 mutation panels for CF carrier testing recommended by the ACMG in 2001 varies by ethnic group.

REPRODUCIBILITY (PRECISION)

Zhang and colleagues,[3] working at Cetus, reasoned that probes bound by UV or heat, were attached in random fashion to the filter and sometimes to each other, making it difficult to determine consistent or optimal hybridization conditions. Until then, Saiki and his collaborators1-4-9-1 had been using nucleotidyl transferase-tailed polyT oligonucleotides under UV radiation to conjugate amino-derivatized membranes (Biodyne B); but Zhang et al. showed that oligonucleotides could be amino-functional-ized by introducing these nucleophiles at 5' oligomer ends during synthesis, and that these would be reactive with charged membranes. After attachment, reactivity of activated carboxyl groups on the membrane remains high; therefore blocking previously activated sites through charge neutralization is necessary before using the membrane in a hybridization reaction, as nucleic acids can otherwise attach to the sites at a low, but significant level, even without an amino-linker. Although charge neutralization and blocking were initially achieved by the Cetus group using hydroxylamine, the UCSF group later found NaOH just as effective.

A Belgian group[10] found that empirical adjustment of the hybridization conditions was a requirement in developing appropriate molecular interrogators for the common Belgian mutations, AF508, G542X, and N1303K. Cuppens et al. also showed that a multiplex PCR was a requirement for detecting the desired variety of mutations at the CF locus. Both of these important observations, the requirement for multiplex PCR and that for reproducible, empirically derived hybridization probes and conditions, have become understood as necessary prerequisites for a successful RDB.

Zhang et al.[3] also emphasized the importance of the physical relationship between the interrogated oligonu-cleotide and the solid support surface through the use of spacer linkers. In controlled experiments, they found that steric spacers aided hybridization and that oligonucleo-tides without linkers were up to fourfold less efficient in hybridization (see Ref. [1] for details).

Despite the development of covalent attachment by the predecessor Cetus group, the Roche Molecular Systems progeny group developed a proprietary process for the manufacture of BSA-conjugated oligonucleotides. The process involves the synthesis of 5'-amino link oligonu-cleotides followed by incubation with BSA and coating onto nylon membranes.[1,11] This proprietary process is used by Roche for the manufacture of their Linear Array Panel (LAp).

Commercialization of RLB hybridization strips required significant effort in ensuring their reproducibility and uniformity. During the period of scale-up at Roche Molecular Systems, it was found that dot blots themselves often gave equivocal results in the form of a halo around the dots. This had more to do with surface tension considerations when applying the amino-conjugated or BSA-conjugated oligos (''printing'') than it had to do with any other consideration. As a consequence, a decision was made to convert the dots to lines of uniform width. Roche developed its own in-house technology for printing line probes, although off-the-shelf commercial technology is available.[1] Extensive improvements in reproducibility and manufacture accompanied the commercialization process. Incubation temperature, temperature equilibration, and uniformity in conjugate and substrate distribution are known to be critical variables for color development. Optimum conditions for nucleo-tide hybridization and development of the RDB strips were devised and automated by Roche and Innogenetics, Inc., through the use of Tecan's Profiblot and Dynal's AutoReli. These shaking baths give uniformity to the strip development that is not easily achieved in any other fashion.

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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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