Abstract: A triple host vector is described which is capable of expressing an inserted protein coding DNA sequence in any of three distinct host systems. The vector includes three promoters, one each for vertebrate, bacterial and insect host cells, so that the protein coding sequence can be cloned into the vector once and the vector can be used to express protein in all three types of host cells. The arrangement and selection of the promoters contributes to expression characteristics in all three host systems that is comparable to single host expression vectors.

Abstract: A method for performing coupled in vitro transcription and translation reactions is disclosed. In the transcription reaction, the quantity of mRNA produced is limited to a level of less than about 2.5 micrograms in a 10 microliter volume prior to the translation elements being added. Limiting the level of mRNA produced prevents saturation of the translational processes and thus aids in the efficiency and fidelity of the translation process.

Abstract: A method for performing coupled in vitro transcription and translation reactions is disclosed In the transcription reaction, the quantity of mRNA produced is limited to a level of less than about 2.5 micrograms in a 10 microliter volume prior to the translation elements being added. Limiting the level of mRNA produced prevents saturation of the translational processes and thus aids in the efficiency and fidelity of the translation process.

Abstract: A vector for the direct cloning of the products of PCR protocol incorporates single nucleotide overhangs at one or both ends of a linearized DNA segment. The single nucleotide overhangs are uracil or inosine residues, as desired, to facilitate cloning of the desired PCR products.

Abstract: A method for inactivating, at a desired stage of an in vitro process, a target enzyme having coupled thereto a biotin molecule, includes adding to a reaction mix an inactivating protein having an affinity for the biotin molecule that is sufficient to inhibit the activity of the target enzyme.The method is embodied in the ribonuclease activity of the enzyme RNase S, which can be active in a form composed of an S peptide and an S protein, not covalently bound together, which associate to form the catalytic molecule. By adding an affinity moeity to the S peptide, it is possible to a second, inactivating protein having affinity for the affinity moeity to disassociate the S peptide from the S protein, and thereby terminate catalytic activity of RNase S at a desired point in any reaction.

Abstract: A method for forming directionally clonable randomly primed cDNA molecules includes the step of priming first strand synthesis using a set of first strand cDNA primers having the sequence 5'-XXNNNNNN-3' where XX is a constant dinucleotide pair across the set and NNNNNN is a random hexanucleotide, the set including primers representing all random hexanucleotides.

Abstract: A method for determining the nucleic acid sequence that encodes ligand binding domain on any protein encoded by a known gene includes the steps of non-specifically cleaving the coding region of the known gene, size-selecting the cleavage products, cloning the size-selected gene products into an expression vector active in a heterologous host, using a novel cloning strategy expressing the peptides produced by the clones and screening for clones that produce peptides that interact with an antibody or ligand of interest.