Roche Diagnostics Operations, Inc.

Roche Diagnostics Gmbh

Roche Molecular Systems, Inc.

Boehringer Mannheim Gmbh

F. Hoffmann-la Roche Ag

Waltraud Ankenbauer

Frank Laue

Dieter Heindl

Christine Ebenbichler

Harald Sobek

Gudrun Schmitz-Agheguian

Vitaly Svetlichny

Elizaveta Bonch-Osmolovskaya

Bernhard Angerer

Ursula Markau

Bruno Frey

Michael Greif

Dietmar Schomburg

Hans-Peter Josel

Thomas Meier

Rainer Schmuck

Thomas Froehlich

Stefanie Froehner

Gunthar Achhammer

Renate Kolb

Britta Villbrandt

Astrid Reiser

Gudrun Schmitz

Bernd Schneidinger

Christian Weilke

Ulrike Dietrich-veenstra

Stephanie Wessner

Yvonne Maerz

Ursula Grepl

Andreas Huber

Eva Walter

Jessica Strey

Gisela Betzl

Annette Deufel

Rita Haerteis

Ulrike Dietrich-Veenstra

The present disclosure is directed to the stabilization of nucleated cells in a whole blood sample ex-vivo, effected by an additive being a liquid composition for stabilizing an analyte in intact nucleated cells of a whole-blood sample ex-vivo, the composition being an aqueous solution, the solution comprising an anticoagulant, a phosphate salt, a cell-metabolizable sugar, adenine, and an antioxidant, wherein the antioxidant comprises a mitochondria-targeted antioxidant, preferably a mitochondria-targeted antioxidant selected from the group consisting of SkQ1, MitoQ, SS-31, and a mixture thereof. In a specific embodiment, the liquid composition further comprises a protease inhibitor. Further provided is advantageous use of the composition for stabilizing an analyte selected from DNA, RNA and protein in intact nucleated cells of the whole-blood sample ex-vivo, as well as kits including the composition for practicing said use. Additionally, specific methods are provided for stabilizing intact nucleated cells of a whole-blood sample ex-vivo.

Stabilization of whole blood at room temperature

The present invention provides kits and methods for composition ratio control based on dyes that are designed to enable energy transfer between each other. In more detail, with the method of the present invention it is possible to verify the mixing ratio of a first component comprising a first dye with a second component comprising a second dye.

Dye composition for liquid transfer control

The present invention is directed to a composition comprising a DNA Polymerase which is preferably thermostable, Deoxynucleotides, at least one primer oligonucleotide or a pair of amplification primers, and randomized 5-8 mer oligonucleotide, characterized in that said oligonucleotide comprises a modification with an organic hydrophobic moiety Such a composition is specifically useful for performing hot start PCR.

Nucleic acid amplification in the presence of modified randomers

The present invention is directed to a method for performing a one-step RT-PCR for amplifying a target RNA comprising the steps (i) providing a sample which is supposed to contain said target RNA (ii) adding a reaction mixture comprising all reagents necessary to reverse transcribe said target RNA into cDNA and amplify at least a portion of said cDNA (iii) incubating said sample for a time interval of 0 seconds to 40 seconds at a temperature between 20&#xb0; C. and 65&#xb0; C., and (iv) subjecting said sample to multiple cycles of a thermocycling protocol wherein the temperature of said sample is varied between at least a first temperature between 37&#xb0; C. and 72&#xb0; C. and a second temperature between 85&#xb0; C. and 100&#xb0; C.

Rapid one-step reverse transcriptase PCR

The present invention is directed to a synthetic peptide having a length of not more than 30 amino acids comprising a divalent cation binding site. Such a peptide according to the present invention is part of a composition for nucleic acid amplification and provides for a so-called hot start effect.

PCR hot start by magnesium sequestration

A purified thermostable enzyme is derived form the thermophilic archaebacterium. The enzyme can be native or recombinant, retains approximately 90% of its activity after incubation for two hours at 95&#xb0; C. in the presence of stabilizing agents and possesses 3'-5&#x2032; proofreading exonuclease activity. Thermostable DNA polymerases are useful in many recombinant DNA techniques, especially nucleic acid amplification by the polymerase chain reaction (PCR).

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It was found that a fragment of nativeDNA polymerase (TaqWT) lacking 288 N-terminal amino acids (Taq&#x394;288) possesses an increased thermostability over TaqWT, Taq&#x394;279, and Taq&#x394;289. The present invention therefore provides Taq&#x394;288, recombinant expression vectors encoding the same or derivatives thereof, as well as purification protocols for Taq&#x394;288. The invention also encompasses kits containing Taq&#x394;288 as well as the use of Taq&#x394;288 and kits containing Taq&#x394;288. In addition, the invention encompasses methods for the sequencing a nucleic acid template and methods for amplifying a target nucleic acid.

Thermostable Taq polymerase fragment

The present invention is directed to a novel chemical compound comprising the structure [Xx-(CH2)m-phosphate-Yy]n, characterized in that 3&#x2266;m&#x2266;6, 30&#x2266;n&#x2266;60, each x and y is independently from each other 0 or 1, each X and Y is independently from each other any photometrically measurable entity; provided that the terminal X can also be an &#x2014;OH group or a phosphate group, and further provided that the terminal Y can also be an &#x2014;OH group. Such a compound can be used as a suitable hot start additive for PCR based amplification of nucleic acids.

Polyanion for improved nucleic acid amplification

A purified thermostable enzyme is derived from the thermophilic archaebacteriumThe enzyme can be native or recombinant, retains approximately 90% of its activity after incubation for two hours at 95&#xb0; C. in the presence of stabilizing agents and possesses 3'-5&#x2032; proofreading exonuclease activity. Thermostable DNA polymerases are useful in many recombinant DNA techniques, especially nucleic acid amplification by the polymerase chain reaction (PCR).

A purified thermostable enzyme is derived form the thermophilic archaebacterium. The enzyme can be native or recombinant, is stable under PCR conditions and exhibits double strand specific exonuclease activity. It is a 3'-5&#x2032; exonuclease and cleaves to produce 5&#x2032;-mononucleotides. Thermostable exonucleases are useful in many recombinant DNA techniques, in combination with a thermostable DNA polymerase like Taq especially for nucleic acid amplification by the polymerase chain reaction (PCR).

Thermostable enzyme promoting the fidelity of thermostable DNA polymerases&#x2014;for improvement of nucleic acid synthesis and amplification in vitro

The invention concerns polymerase chimeras which are composed of amino acid fragments representing domains and which combine properties of naturally occurring polymerases that are advantageous with regard to a particular application. It has surprisingly turned out that the domains from the various enzymes are active in the chimeras and exhibit cooperative behavior. In addition the present invention concerns a process for the production of the chimeras according to the invention and the use of these chimeras for the synthesis of nucleic acids e.g. during a polymerase chain reaction. Moreover the present invention concerns a kit which contains the polymerase chimeras according to the invention.

Polymerase chimeras

The present invention is directed to a new composition for performing a nucleic acid amplification reaction comprising (i) a thermostable DNA-Polymerase, (ii) a thermostable 3'-5&#x2032; Exonuclease, and (iii) at least one primer for nucleic acid amplification with a modified 3&#x2032; terminal residue which is not elongated by said thermostable DNA-Polymerase as well as methods for performing a PCR reaction using this composition. Furthermore, the method is directed to kits comprising such a composition.

Composition and method for hot start nucleic acid amplification

A purified thermostable enzyme is derived from the thermophilic archaebacterium. The enzyme can be native or recombinant, is stable under PCR conditions and exhibits double strand specific exonuclease activity. It is a 3'&#x2013;5&#x2032; exonuclease and cleaves to produce 5&#x2032;-mononucleotides. Thermostable exonucleases are useful in many recombinant DNA techniques, in combination with a thermostable DNA polymerase like Tag especially for nucleic acid amplification by the polymerase chain reaction (PCR).

Thermostable enzyme promoting the fidelity of thermostable DNA polymerases-for improvement of nucleic acid synthesis and amplification in vitro

A purified DNA polymerase exhibiting reverse transcriptase activity in the presence of magnesium ions and/or manganese ions having reduced or no 5&prime;-3&prime;-exonuclease activity and substantially no RNaseH activity and obtainable from

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Penzberg, Germany

Waltraud Ankenbauer