The Perkin-Elmer Corporation

Other

Hoffmann-La Roche Inc.

Cetus Corporation

The State University of New York

University of Oregon

Cetus Corportion

Will Bloch

Timothy M Woudenberg

Lawrence A Haff

Enrico G Picozza

Steven Menchen

David H Tracy

Robert P Ragusa

Paul G Saviano

Ben F Johnson

Jonathan C Raymond

Joseph DiCesare

Alan R Read

Patrick J Sheridan

Richard W Noreiks

Robert J Goodson

Robert J Drummond

Gerard J Nuovo

Brian W Matthews

Pamela L Toy

A capillary electrophoresis system and method of electrokinetically loading a capillary electrophoresis sample into a separation medium in a capillary tube in which an entangled polymer matrix is formed having the sample embedded therein. The matrix has a mesh size effective to retard movement of macromolecules such as DNA sequencing templates through the matrix when an electric field is applied across the matrix. The entangled polymer matrix is formed by a linear polymer having a molecular weight of at least 20K Daltons. Furthermore, the invention includes stable denaturants useful for the electrophoresis of nucleic acids.

Entrapment of nucleic acid sequencing template in sample mixtures by

Apparatus and method for performing a nucleic acid amplification reaction and preferably a polymerase chain reaction (PCR) in a reaction mixture in at least one capillary tube. Several different embodiments are disclosed. One embodiment cycles a sample through a capillary tube loop passing through two thermostatted fluid baths. Another embodiment has the capillary tube routed alternatingly between two heat exchangers to that the sample makes only one pass through the tube. Other embodiments maintain the heat exchangers stationary and translate the samples between them. Still further embodiments maintain the samples stationary and either automatically translate or rotate the heat exchangers past the samples contained within the capillary tubes to perform the thermal cycles necessary for the amplification reaction.

Nucleic acid amplification reaction apparatus and method

Improved methods, compositions and kits for detecting mutations in nucleic acids are disclosed, such comprise annealing a piece of control nucleic acid without mutations to a piece of test nucleic acid very similar in sequence to the control nucleic acid but possibly containing mutations, treating this mixture with potassium permanganate or hydroxylamine to remove mismatched bases from the duplex nucleic acid, treating the resulting nucleic acid with any of a class of diamines, triamines, and tetraamines analogous to 1,2-ethylenediamine to cleave abasic sites, and then analyzing the chemically treated nucleic acid to determine whether cleavage has occurred and approximately at what position in the nucleic acid any cleavage has occurred. Inclusion of betaine in the base-removal reactions improves their sensitivity and specificity. This series of non-hazardous, non-noxious, chemical treatments can be performed rapidly in a single reaction tube with no or minimal intervening separation steps; and the cleavage product can be prepared for the analysis step without removing the chemical reagents used to effect cleavage.

Detection of mutations in nucleic acids by chemical cleavage

Solvents for salt-gradient anion-exchange separation of nucleic acids, especially double-stranded DNA and especially by liquid chromatography, are improved by replacing NaCl as the eluting salt with any of a wide range of alkyl ammonium salts and can be used to elute nucleic acids in strict order of increasing length, with reduced sensitivity to elution temperature and salt concentration. Anion-exchange chromatography with these solvents is well suited for identification of DNA fragments on the basis of size, with greater accuracy, precision, and resolvable size range than often is possible with gel electrophoresis.

Precision and accuracy of anion-exchange separation of nucleic acids

Nucleic acid amplification reaction apparatus

Improvements to the polymerase chain reaction (PCR), a process for in vitro enzymatic amplification of specific nucleic acid sequences, can be achieved by changing the way that PCR reagents are mixed and the enzymatic reaction is started and by the replacement of mineral oil, commonly used as a vapor barrier to minimize solvent evaporation, by a grease or wax. The use of such mixtures allows for the delay of reagent mixing until the first heating step of a PCR amplification, thereby reducing the enzymatic generation of nonspecific products which occurs when a complete mixture of PCR reagents, with or without test sample, stands at room temperature or below. These mixtures increase the shelf-life of PCR reagents and increase protection of the laboratory environment against contamination by PCR product.

Compositions and methods for inhibiting dimerization of primers during

Improvements to the in situ polymerase chain reaction (PCR), a process of in vitro enzymatic amplification of specific nucleic acid sequences within the cells where they originate, can be achieved by changing the way that the enzymatic reaction is started. Reaction initiation is delayed until the start of PCR thermal cycling, either by withholding a subset of PCR reagents from the cellular preparation until the preparation has been heated to 50.degree. C. to 80.degree. C., immediately before thermal cycling is begun, or by adding to the PCR reagents a single-stranded DNA binding protein which blocks reaction at temperatures below about 50.degree. C. If the in situ PCR is performed on cellular preparations already attached to a microscope slide, thermal cycling also is facilitated by use of a thermal cycler sample block or compartment designed optimally to hold the microscope slide and any vapor barrier covering the slide.

In situ polymerase chain reaction

Use of grease or wax in the polymerase chain reaction

Xylose isomerase (XI) muteins useful in the conversion of glucose to fructose or xylose to xylulose are obtained in usable amounts by protein structural and recombinant DNA methods, including x-ray crystallography, cloning, computer graphic modeling and site-directed mutagenesis and expression of the bacterial DNA sequences encoding native procaryotic xylose isomerase. These native sequences are altered to encode the xylose isomerase muteins having improved catalytic function and/or thermostability.

Procaryotic xylose isomerase muteins

The invention herein is directed to methods using Procion dyes to perform separations of interest in manipulating the NAD.sup.+ -independent ribotoxins. The methods are useful for preparing therapeutic agents containing these ribotoxins or their A polypeptide components. This separation method has been applied in particular to preparing hybrid toxins containing ricin toxins, both for purifying the resulting products and also for separating the components intended to be used in the preparation of these end products. In addition, a novel ricin isotoxin prepared using the method of the invention is disclosed.

Ribotoxin conjugates

The invention herein is directed to methods using Procion dyes to perform separations of interest in manipulating the NAD.sup.+ -independent ribotoxins. The methods are useful for preparing therapeutic agents contaning these ribotoxins of their A polypeptide components. This separation method has been applied in particular to preparing hybrid toxins containing ricin toxins, both of purifying the resulting products and also for separating the components intended to be used in the preparation of these end products. In addition, a novel ricin isotoxin prepared using the method of the invention is disclosed.

Purification of ribotoxins and their conjugates

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San Mateo, CA, United States of America

Will Bloch