Promega Corporation

The Johns Hopkins University

Paula R Brisco

Rex M Bitner

Steven J Ekenberg

John William Shultz

Craig E Smith

Michelle N Mandrekar

Douglas R Storts

Susanne Selman

Josephine C Grosch

Michelle M Mandreker

Anne Louise Oaklander

Judy Frederiksen

Judy Frederiksen Towne

The present invention relates to methods, kits, and compositions for generating purified RNA samples and purified DNA samples. In particular, the present invention provides methods for generating a purified RNA or DNA sample from a sample containing both DNA and RNA molecules using a binding matrix that preferentially binds DNA or RNA in the presence of an acidic dilution buffer, or using a binding matrix that comprises acid zeolites, as well as compositions and kits for practicing such methods.

Nucleic acid purification with a binding matrix

Solutions containing nucleic acids are treated with an alkaline protease to digest proteins such as nucleases that degrade the nucleic acids. In the isolation of nucleic acids, a biological sample containing nucleic acids is suspended in a solution containing water, buffer and chelating agent, the pH of the solution is adjusted to at least about 10 by adding a solution of sodium hydroxide and anionic detergent, an alkaline protease is incubated in the solution until nucleases are degraded, the pH of the solution is lowered to reduce activity of the alkaline protease by adding a solution having a pH between 3.5 and 4.5 and the alkaline protease is heat inactivated. Lowering of the pH may produce a cloudy solution which is cleared by centrifuging. Nucleic acids are isolated from the cleared solution by alcohol precipitation, or by using paramagnetic particles or a resin matrix containing silica particles. A chaotropic salt can be used to reversibly bind DNA to the resin matrix.

Methods for isolating nucleic acids using alkaline protease

Methods for separating target particles, from a test medium in which the particles are suspended. The method contemplates utilization of blocking or masking particles which block or mask unwanted biological debris which tends to interfere with formation of complexes by which the target particles can be isolated. After centrifugation, the masking particles create a barrier between unwanted debris and test medium containing the target particles which permits the particles to be manipulated or modified without removing them from the debris. In a preferred aspect, magnetic separation techniques are used. In this aspect, non-magnetic 0.04 micron carboxylated polystyrene blocking particles are employed.

High efficiency method for isolating target substances using a

Apparatus and methods for separating magnetically responsive particles from a nonmagnetic test medium in which the magnetically responsive particles are suspended. The separator comprises a container comprising, e.g., plurality of multiwell chambers, which hold the nonmagnetic test medium, one or more pins disposed substantially within the test medium, an external magnet for producing a magnetic field within the test medium. The external magnet is proximate the pins so as to create magnetic flux lines, magnetizing the pins and creating a magnetic field substantially parallel to the longitudinal axis of the pins thereof in the test medium. According to the method of the invention, the container holding the test medium is positioned in the separator, producing a magnetic field operative to cause the magnetically responsive particles to be attracted to and adhere to the pins and which is substantially parallel thereto. The nonmagnetic test medium is separated from the magnetized particles, which may conveniently be washed while adhered to the pins, and subjected to further analysis, preferably while on the pins. The apparatus and method are useful in separating various target substances from test media by means of substances coated on the magnetic particles which bind specifically to the target substance.

Multisample magnetic separation device

Growing community of inventors

Madison, WI, United States of America

Paula R Brisco