Biochemical indicators of muscle growth in the snow crab Chionoecetes opilio (O. Fabricius).

This study examined the relationships between muscle growth rate, the activity of metabolic enzymes and the RNA:DNA ratio, in adult snow crabs Chionoecetes opilio. After moulting, crabs were assigned to three feeding rations to attain a range of tissue growth rates. Muscle growth rate, estimated by the variation in dry tissue content per ml of merus of the first walking leg, was positively correlated with changes in muscle cell number, as evaluated by the DNA content per ml of merus. However, no significant correlation was detected between growth rate and the variation in muscle cell size, the latter being estimated by the change in the protein:DNA ratio. This is due to the fact that, in starved crabs, a reduction in the number of cells is partly compensated by a size increment of the remaining ones. This phenomenon also weakened the overall relationship between muscle growth rate and the phosphofructokinase (PFK) capacity per ml of merus. The simple correlation between those two variables was significantly positive for animals which increased their mass of muscle but insignificant for those which were loosing muscle mass. The lactate dehydrogenase (LDH), citrate synthase (CS) and cytochrome c oxidase (CCO) capacity per ml of merus did not match growth rate. The significant simple correlations that were detected between growth rate and the various enzyme activity expressed per g of protein, per µg of DNA and per g of dry mass did not hold when partial correlations were computed. Variations in muscle cell size were related to adjustments in the quantity of RNA per cell, as depicted by the RNA:DNA ratio. Since muscle growth was not correlated with the variation in muscle cell size, it was not correlated with the RNA:DNA ratio either.

Fast and reliable screening of mutations in human tumors: use of multiple fluorescence-based long linker arm nucleotides assay (mf-LLA).

Human tumor samples were screened for point mutations by adapting a mobility-shift assay to automated DNA sizing. This screen identifies the type of point mutation and relative amount of mutated DNA sequences present in a sample. Test samples having known hypoxanthine-guanine phosphoribosyl transferase (hprt)/exon-3 sequence mutations were characterized by: (i) PCR amplification, (ii) fluorescent dye-primer extension with 36-atom linker derived deoxycytosine or deoxyuridine triphosphate and the remaining three natural nucleotides and (iii) sizing of the resulting fluorescently labeled modified strands, using an automated DNA sequencer. Routinely, a range of sizes is observed among the sequence variants of a single DNA target sequence. This is because nucleotide analogs are incorporated into DNA strands in a sequence-dependent manner, resulting in composition-dependent electrophoretic mobility. Thus, point mutations are identified as shifts in mobility between the fluorescently labeled modified strands of the control and test samples. The twenty different hprt/exon-3 single-base substitution mutations tested were easily identified, even at fourfold dilution with control DNA.

Detection of HIV-1 DNA in cells and tissue by fluorescent in situ 5'-nuclease assay (FISNA).

The critical aspects of successful in situ amplification include fixation, permeabilization, amplification and detection. We address these aspects and present a novel detection scheme that eliminates hybridization following amplification. We use the 5'-nuclease activity of Taq polymerase to cleave in situ a 5'-reporter dye from an oligonucleotide probe which hybridizes to the target amplicon during amplification. The 5'-reporter dye is disassociated from the 3'-quenching dye and remains localized by charge interactions. In addition, we describe probe design constraints for 5'-nuclease assays both in solution and in situ. Using this technique, we show the sensitive and specific detection of HIV-1 DNA in cells lines and tissue from HIV-1-infected individuals.

A new approach to template purification for sequencing applications using paramagnetic particles.

A new method using traditional hybridization methodology, coupled with the new magnetic particle technology, has been developed for DNA purification, specifically for sequencing applications. The method is similar to the reverse hybridization blot system; however, a specific oligonucleotide probe was attached to the paramagnetic particle instead of a sheet membrane. The target DNA containing the complementary sequence of the probe hybridizes to the probe that is attached to the bead and is then magnetically removed from solution, washed and collected. This system eliminates the need of organic extractions and precipitation/concentration steps. The entire hybridization-purification system can be done in a 1.5-ml microcentrifuge tube making the method ideal for automation. M13 phage clones were purified with this method, both by manual means and by using the CATALYST 800 Molecular Biology LabStation fitted with a prototype magnetic station, and then sequenced. DNA sequencing results obtained with this system were reproducible and gave excellent length of read with low background.