Biotechnica International, Inc.

H.j. Heinz Company

Abbott Laboratories Corporation

Omnigene, Inc.

Keith C Backman

John J Carrino

Ramaswamy Balakrishnan

Christiane Munkholm

Randolph T Hatch

Robert R Yocum

Thomas G Laffler

George H Shimer, Jr

Sheila B Bond

The present invention involves methods of improving LCR and PCR amplification schemes by modifying at least one probe/primer end so that the probability of the probe/prirner contributing to spurious signal development is greatly reduced. Only after specific hybridization of the modified probe/primer with true target, are the modified ends 'corrected' in a target dependent fashion to allow participation of the probe/primer in the enzymatic assembly reaction. Specific modifications depend on whether the assembly is done by ligation (as in LCR) or by extension/elongation (as in PCR).

Method and kits for amplifying target nucleic acids applicable to both

The present invention involves methods of improving the Ligase Chain Reaction (LCR.TM.) amplification schemes by modifying at least one probe end so that the probability of the probe contributing to spurious ligation and signal development is greatly reduced. Only after specific hybridization of the modified probe with true target are the modified ends 'corrected' by endonuclease IV in a target dependent fashion to allow participation of the probe in the enzymatic ligation reaction. Specific modifications include 3' phosphate blocking groups and nucleic acid overhangs containing an abasic site at the point of ligation. Further embodiments include probes modified to contain ribonucleotide moieties which, after amplification, can be cleaved by RNase to destroy the amplification products and reduce the risk of contamination.

Ligase chain reaction with endonuclease IV correction and contamination

An optical detection method for detecting specific ligands in an immunoassay is formed by diffracting a beam of light through slits formed in a mask and thererby forming an interference pattern. The diffracted light of one of the slits is distributed by an assay having a ligand which when reacted with anti-ligand changes the optical characteristics of the assay thereby changing the interference pattern in a concentration dependent manner.

Ligand assay using interference modulation

A method of controlling of cell mass by starving cells that are ppc.sup.- and pc.sup.- for tricarboxylic acid cycle (TAC) intermediates, while fermenting a product compound. The cells are adapted to enable or enhance conversion of a raw material to the product via a bioconversion pathway that does not include any TAC intermediates, and preferably they are either conditionally or permanently incapable of producing a net increase in TAC intermediates via any anaplerotic pathways. First the cells are cultured to the desired cell mass under growth medium conditions characterized by the presence of at least one TAC intermediate, or by the presence of a compound that the cells can convert to a TAC intermediate via an anaplerotic pathway under the conditions present in the growth medium. In that way the medium provides the cell enough TAC intermediates to support growth. When the desired cell density has been reached, the cells are cultured under product-production medium conditions in which the cell does not receive adequate TAC intermediates to support growth, because the medium lacks TAC intermediates as well as compounds that they all can convert to TAC intermediate via an anapleortic pathway.

Method of biosynthesis of phenylalanine

A method of continuous product formation using at least two continuous fermentation units and a microorganism capable of being induced, in response to environmental conditions, to undergo a genetic alteration from a state favoring microorganism growth to a state favoring product production by the microorganism. The first continuous fermentation unit is maintained at environmental conditions selected to favor growth of the microorganism and to be nonpermissive for the genetic alteration. The microorganism is grown continuously in the first unit, and a portion of the growing microorganism cell mass is transferred via connecting means to the second continuous fermentation unit. Either the connecting means or the second unit is maintained at second environmental conditions selected to effect the genetic alteration. The altered microorganism is cultured in the second unit. Exudate from this second fermentor (containing microorganism mass and medium) is continually removed and the product which is present, either in the microorganisms themselves or the medium surrounding them, is extracted.

Dual-stage fermentation

An expression vector having two structural genes that form a synthetic operon expressed under the control of a single regulatory sequence. The operon genes correspond to the structural component of naturally occurring genes whose expression is controlled by distinct separate regulatory sequences. The operon genes code for enzymes in a biosynthetic pathway for producing a desired compound, and at least one of those operon genes is feedback derepressed. The vector is used to transform host cells that are cultured to produce the desired product.

Method of biosynthesis and cells therefor

Proteins having chorismate mutase-prephenate dehydratase (CMPD) activity, but lacking phenylalanine sensitivity are produced by genetic engineering. The proteins contain a sequence substantially corresponding to the N-terminal 337 amino acids of Escherichia coli CMPD. Expression vectors including genes coding for those proteins and regulatory DNA enabling their expression are used to transform host microorganisms, which are cultured to produce phenylalanine.

Enzyme deregulation

An Escherichia coli chromosomal DNA segment is engineered for controllable excision and loss from the E. coli cell population. The DNA segment has a gene determining a function that is lost in the absence of that chromosomal segment. Specifically, the segment includes a pair of lambdoid phage att sites positioned at opposite ends thereof, and a gene encoding the function. The E. coli cell includes DNA encoding lambdoid phage int and xis, positioned for transcription under control of an external stimulus. By excising the chromosomal DNA segment once a desired cell population is achieved, cell growth and diversion of intermediates from a desired fermentation product are diminished or avoided.

Controlled gene excision

In vivo regulation of protein production is achieved by rearranging DNA segments comprising a protein-producing gene (i.e., protein-encoding DNA as well as regulatory DNA to effect the expression of the protein-encoding DNA in the host), in response to a change in an environmental condition such as temperature. The rearrangement is synchronized and directional (irreversible) in members of the cell population, because it is catalyzed by a lambda phage site-specific recombination enzyme system that operates on a pair of lambda phage attachment sites to rapidly drive the rearrangement and to avoid the reverse reaction. The cells include means to produce the lambda enzyme system in response to the change in environmental condition. By engineering one of the attachment sites within the gene that produces the protein whose production is to be regulated (yielding two gene segments), the synchronized rearrangement operates to change the gene from one configuration to another. In only one of these configurations are the gene segments positioned and oriented for protein production. Specifically, the protein-producing configuration is: gene segment one-attachment site-gene segment two. The attachment site is exogeneous to the gene, i.e, it does not occur in that location naturally and is positioned there by engineering techniques. The regulated protein production is useful, e.g., in fermenting the desired product, by allowing cell growth to proceed in the absence of product formation. When the desired cell mass is achieved, product production is enabled by the rearrangement.

Growing community of inventors

Bedford, MA, United States of America

Keith C Backman