Promega Corporation

Douglas R Storts

Poncho Meisenheimer

Wenhui Zhou

Min Zhou

Kimberly K Knoche

Leopoldo G Mendoza

John William Shultz

James Robert Hartnett

Paul Otto

Michael R Slater

Craig E Smith

Fen Huang

Susanne Selman

Josephine C Grosch

Paula R Brisco

Katharine M Miller

Alexander Novikov

Judy Frederiksen

Elena Bolchakova

Galina A Velikodvorskaya

Judy Frederiksen Towne

The present disclosure includes compositions and methods for improved DNA amplification reactions. In particular, the present disclosure provides compositions and methods for hot-start PCR applications using DNA polymerase inhibitors that minimize non-specific DNA amplification by inactivating DNA polymerase at lower temperatures.

Polymerase inhibitors and related compositions and methods

The present invention relates to compositions of thermostable DNA polymerases derived from the hyperthermophilic eubacteria. In particular, the present invention comprises thermostable DNA polymerases from the hyperthermophilic eubacterium known as Thermotoga neapolitana. The present invention provides methods for utilizing naturally-occurring and non-naturally-occurring forms of T. neopolitana DNA polymerase. The T. neopolitana DNA polymerases of the present invention are used in combination with other compounds, including but not limited to pyrophosphatase and DNA polymerases from other thermophilic or hyperthermophilic organisms.

Thermophilic DNA polymerases from Thermotoga neapolitana

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

A new process and kit are described that combines methods for generating the nucleotide base sequence of a DNA molecule with an ultra-sensitive silver staining protocol. This new combination of technologies allows for a direct, non-instrument based visualization of electrophoretically separated sequencing fragments. This non-radioactive system includes sequencing the DNA molecule by forming a set of fragments using an enzymatic dideoxy-mediated chain termination method, electrophoretically separating the DNA fragments on a gel medium, and exposing the gel medium to ultra-sensitive silver-staining solutions for a time determined by viewing the silver stain reacted primer extension products.

Non-radioactive DNA sequencing

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Madison, WI, United States of America

Douglas R Storts