Codon-based mutagenesis using dimer-phosphoramidites.

A new approach for the synthesis of randomized DNA sequences containing the 20 codons corresponding to all natural amino acids is described. The strategy is based on the use of dinucleotide phosphoramidite building blocks within a resin-splitting procedure. Through this protocol, a minimal number of seven dimers is sufficient to encode all 20 natural amino acids. This synthesis procedure is extremely flexible and allows codon usage from different hosts to be accommodated.     

Bioseparation using affinity techniques.

Bioseparation of proteins from dilute solutions using different novel affinity procedures is reviewed. Emphasis is also placed on the quality of the product separated. Whenever possible, physical insights into the separation procedure are provided, besides indicating suitable directions where appropriate further research may be carried out. The procedures analyzed are different affinity chromatographic techniques, affinity separation using liquid perfluorocarbon supports, water soluble nonionic surfactants for affinity bioseparations, affinity cross-flow filtration, bioaffinity separation using reversed micelles, affinity precipitation and dual-functional affinity protein purification.     

Histone propinquity using imidoesters.

Native chromatin was reacted with bifunctional protein cross-linking reagents of varying molecular sizes. Short cross-linkers do not produce significant yields of polymeric histones whereas intermediate-sized materials such as dimethyladipimidate produce extensive interaction between histones. Dimethylsuberimidate reacts with all histone fractions but only generates polymers of F₁ and of F_2b and F_2a2.     

Immunoaffinity chromatography using electroelution.

Immunoaffinity chromatography and electroelution of protein from solid-phase matrices are two powerful tools often used to purify proteins. In this study, we combined these two techniques and found that antigen was effectively recovered from immunoaffinity resins by electroelution. Yields ranged from 90.5% to 62.8%, with a mean of 74.0 +/- 7.4% (mean +/- standard deviation). A major portion of the eluate, 79.4 +/- 13.1%, was concentrated in a volume of 100 microliters and 94.0 +/- 2.0% was recovered in 200 microliters, even when 1 ml of resin was used. Electroelution had no major effect on the electrophoretic mobility of the antigen. Two distinct antigens, a relatively hydrophilic 230-kDa protein and a hydrophobic 28-kDa protein were successfully electroeluted. In addition two types of immunoaffinity resins, monoclonal antibody covalently linked to CNBr-activated Sepharose or immobilized protein A, were found to be compatible with this method.     

Subcloning using simplified adaptor addition.

A simplified procedure for the addition of synthetic oligonucleotide adaptors to subclone DNA fragments with incompatible ends is presented. An organophosphate degradation gene on a PstI fragment was cloned into the HindIII site of the fungal vector pH1S. The opd gene specifies parathion hydrolase and was first isolated from a Flavobacterium sp. The gene was present in 12% of the plasmids recovered and was inserted in either direction with similar frequencies: 53% with the opd start codon distal to the single SalI site of pH1S and 47% in the other orientation. All enzymatic steps were carried out in a single microconcentrator eliminating DNA loss through manipulation and transfer. Normally, during adaptor or linker addition, a larger number of oligonucleotides are attached at each end of the insert DNA and must be removed before cloning. The need for enzymatic digestion to remove excess adaptors was avoided. Traditional methods have utilized phenol/chloroform extraction, ethanol precipitation, gel filtration chromatography, spermine precipitation, or preparative gel electrophoresis. Eliminating these steps resulted in a simpler, more reliable procedure.     

Electrode-based immunoassays using urease conjugates.

Urease conjugates are employed for competitive-binding enzyme immunoassays (EIA) of a model antigen, bovine serum albumin (BSA), and of cyclic AMP (cAMP). Urease activity bound to a double-antibody solid phase is determined with an ammonia gassensing electrode, after appropriate washing steps. Cyclic AMP analogs coupled to urease are used to determine their effect on the overall response characteristics of the cAMP assay. The use of urease as a label for EIA purposes is shown to yield sensitive assays for both proteins (BSA < 10 ng/ml) and haptens (cAMP < 10^?8m) with good day-to-day reproducibility.     

Allergy screening using a microcomputer.

Data are presented to show that a microcomputer can be programmed to: (1) analyse a standard allergy questionnaire, (2) reliably predict the ranked order probabilities with which IgE antibody tests will produce positive results, (3) store the IgE test results, and (4) print a comprehensive report that summarises and integrates the clinical and laboratory data. Consequently, the practitioner who refers a blood sample and a questionnaire completed by the patient to a centre where both can be analysed will obtain enough practical information to decide whether to treat or refer that patient to a specialist. The microcomputer is therefore potentially of great value in any preliminary allergy investigation.     

Distillery effluent treatment using Artemia.

Artemia functions as living filter for the secondary treatment of distillery effluent, which reduces up to 69% of the total solids and 33.34% of BOD in the saline medium of 60 ppt. Chemical analysis of diluted effluent after treatment with Artemia showed a significant (P less than 0.001) decrease in the electrical conductivity, calcium and potassium values. However, an increase in sodium concentration and pH values, was recorded during experiment.     

Studying DNA-protein interactions using NMR.

NMR spectroscopy is emerging as a powerful tool in molecular biology and biotechnology; one aspect of which is the use of one- and two-dimensional NMR methodologies to investigate the interactions of proteins with DNA. The dynamic and structural information which NMR can provide, on the changes in conformation and molecular flexibility, complements X-ray crystallography data and enables mechanistic models of DNA-protein interactions to be formulated.     

Model-free linkage analysis using likelihoods.

Misspecification of transmission model parameters can produce artifactually negative lod scores at small recombination fractions and in multipoint analysis. To avoid this problem, we have tried to devise a test that aims to detect a genetic effect at a particular locus, rather than attempting to estimate the map position of a locus with specified effect. Maximizing likelihoods over transmission model parameters, as well as linkage parameters, can produce seriously biased parameter estimates and so yield tests that lack power for the detection of linkage. However, constraining the transmission model parameters to produce the correct population prevalence largely avoids this problem. For computational convenience, we recommend that the likelihoods under linkage and non-linkage are independently maximized over a limited set of transmission models, ranging from Mendelian dominant to null effect and from null effect to Mendelian recessive. In order to test for a genetic effect at a given map position, the likelihood under linkage is maximized over admixture, the proportion of families linked. Application to simulated data for a wide range of transmission models in both affected sib pairs and pedigrees demonstrates that the new method is well behaved under the null hypothesis and provides a powerful test for linkage when it is present. This test requires no specification of transmission model parameters, apart from an approximate estimate of the population prevalence. It can be applied equally to sib pairs and pedigrees, and, since it does not diminish the lod score at test positions very close to a marker, it is suitable for application to multipoint data.