Akzo Nobel N.v.

Primagen Holding B.v.

Bob Van Gemen

Eveline Catherina A C Timmermans

Hans H Liao

Tim Kievits

Peter Franklin Lens

Marinus Petrus De Baar

Akio Sugiyama

Caroline Louise Ovyn

Antoinette C Van Der Kuyl

Adriana Frederieke Schukkink

John Dekker

Jing Zhang

Dianne Arnoldina Van Strijp

Jolanda Maas

Harmannus Bernardus Van Schijndel

Anthonij De Ronde

Michel Mathijs Klerks

Dianne Arnoldina Margaretha Van Strijp

Irene Johanna M Dobbelaer

Adriana Fredericke Schukkink

The invention relates to the diagnosis of disease or the determination of functioning of cellular organisms, being of multi-cellular or unicellular nature, being visible by the naked eye or being a microorganism. The invention provides a method for determining functioning of a cellular organism comprising determining the relative ratio of a first endosymbiont cellular organelle nucleic acid and/or gene product thereof in a sample obtained from the organism in relation to the amount of a second nucleic acid and/or gene product thereof.

Method of determining therapeutic activity and/or possible side-effects of a medicament

Methods for amplifying nucleic acid in a sample comprising providing the sample with a set of primers to enable synthesis of at least one nucleic acid strand complementary to at least part of the nucleic acid, wherein the set of primers comprises between 3-8 random bases, preferably clustered near the 3&prime; end of each primer in said set of primers. The methods of the invention are useful, for example, for determining whether samples derived from humans, mammals, poultry, or fish comprise nucleic acid of a pathogen. The methods are further suited for typing the pathogen and typing particular variants of said pathogen. The methods are also suited for the elucidation of the gene expression profile or genetic profile of cells.

Universal nucleic acid amplification system for nucleic acids in a sample

The invention provides a method for reducing background in hybridization reactions of nucleic acids involving at least two homologous probes, wherein at least one of the probes is non-linear, or two homologous target sequences and a non-linear probe. Background is reduced by introducing an intended mismatch with a target sequence in at least one of the probes. The presence of the mismatch reduces the specificity of probes not entirely complementary to a target sequence to such an extent that the background signal is reduced. A set of mixed homologous probes, wherein at least one of the probes is non-linear, comprising such specific mismatch is also provided. The set can be used for the detection of variants of a family of nucleic acids, for instance a number of HIV variants. The invention also provides kits for carrying out the methods according to the invention.

Reducing background in hybridization reactions

The present application relates to mutated RNA polymerases from bacteriophages that have increased stability, for example under high temperature conditions. One example of bacteriophage encoded RNA polymerase is the T7 RNA polymerase. T7 is a bacteriophage capable of infecting cells. Examples of other infecting T7-like bacteriophages are T3, &oslash;I, &oslash;II, W31, H, Y, A1, croC21, C22 and C23. An example of a infecting bacteriophage is SP6. The present invention is concerned with the RNA polymerases of T7-like bacteriophages that have been mutated. Due to these mutations the RNAP's have an increased stability. Preferred mutated RNA polymerases according to the invention are mutant RNA polymerases from T7 or SP3 bacteriophages. Due to the high homology between these enzymes, mutations in the T7 gene 1 sequence are likely to have the same effect in the corresponding gene sequence of the T3 bacteriophage. An especially preferred embodiment of the present invention is a T7 RNA polymerase with a serine to proline amino acid change in the protein at position 633 of the amino acid sequence. Since there is 80% homology between the T7 RNA polymerase and the T3 RNA polymerase the same effects of the 633 serine&rarr;proline mutation in the T7 gene may be expected for a 634 serine&rarr;proline amino acid mutation in the T3 RNA polymerase.

Mutant of RNA polymerases with increased stability

The present invention relates to a method for isolating nucleic acid from a sample. Known methods for the isolation of nucleic acid from these materials may comprise the use of a chaotropic substance, for example to lyse cells present in the starting material, and free the nucleic acid therefrom. When a nucleic acid containing material is treated with a chaotrope and contacted with a nucleic acid binding solid phase, the nucleic acid will bind to the solid phase in the presence of the chaotrope and the solid phase with the nucleic acid bound thereto can thus be separated from the remainder of the sample. The method of the present invention for the isolation of nucleic acid from nucleic acid containing starting material is a method wherein the starting material is contacted with a chaotropic substance and, either simultaneously or subsequently, with a nucleic acid binding solid phase, where after the nucleic acid binding solid phase is subjected to a washing procedure and, optionally, the nucleic acid is eluted from the nucleic acid binding solid phase, characterized in that the washing procedure comprises the steps of washing the solid phase with subsequently ode ore more high salt buffer solution(s), optionally an alcoholic solution and a low salt buffer solution.

Method for the isolation of nucleic acid

The present invention is directed to a transcription based amplification method for the amplification of DNA targets. With the method of the present invention an isothermal transcription based amplification method is provided for the amplification of double stranded DNA. The method of the present invention for the amplification of double stranded DNA comprises the steps of contacting said double stranded DNA with at least one oligonucleotide comprising a sequence complementary to a part of the first of the two DNA strands comprised in the double stranded DNA, said oligonucleotide further comprising the sequence of a promoter recognized by a RNA polymerase; a further olignucleotide comprising a sequence complementary to a part of the second strand comprised in the double stranded DNA; an enzyme having RNA dependent DNA polymerase activity; an enzyme having DNA dependent DNA polymerase activity; an enzyme having RNase H activity; an enzyme having RNA polymerase activity, and maintaining the thus created reaction mixture under the appropriate conditions for a sufficient amount of time for the amplification to take place. The method according to the invention is particularly useful for amplifying small DNA molecules, e.g. plasmid DNA. Surprisingly, transcription based amplification can start from double stranded DNA, without the need to treat the DNA with restriction enzymes or, what is more important, to separate the strands before hand by applying heat. All reagents conventionally used with isothermal transcription based amplification reactions can simply be used on starting material containing double stranded DNA, as if it where single stranded RNA. With the present invention it has now been found that essentially the same protocols can be used for isothermal transcription based amplification of double stranded DNA. The method is particularly useful for the detection of small DNA molecules of pathogenic microorganisms enabling diagnosis. In particular the circular HIV-1 DNA molecules that are formed during the replication of the HIV-1 virus can be detected by this method. Detection of such circular HIV-1 DNA molecules indicates active replication of the virus, which can be correlated with disease progression, i.e. development of AIDS.

Transcription based amplification of double stranded DNA targets

Primers and probes for the amplification, detection and typing of

Disclosed is a method of quantifying a target nucleic acid in a test sample by adding to the test sample a known number of molecules of a corresponding nucleic acid comprising a well-defined mutant sequence. Said mutant sequence being discriminatory from the target nucleic acid. Subsequently a competitive amplification reaction of the nucleic acid is performed after which quantification of the amplified nucleic acid is performed by a differential detection.

Quantification of nucleic acid

A process for making the product of a nucleic acid amplification reaction, which amplification reaction employs one or more primer pairs, incapable of being a target for further amplification comprising: contacting the amplified product with an oligonucleotide capable of hybridizing to a stretch a nucleotides of the amplified product, said stretch being situated between the hybridisation sites of a pair of primers, under conditions which allow formation of a hybridisation complex between the oligonucleotide and the amplified product to occur, said oligonucleotide being modified in such a way that it.protects the double stranded part of the hybridisation complex hybridized to the oligonucleotide from degradation, and subjecting the hybridisation complex to a degradative treatment under circumstances such that at least the part of the hybridisation complex capable of hybridizing to the primers is degraded. An assay for diagnosing the presence of a nucleic acid sequence in a sample comprising carrying out the above-mentioned method and a kit for carrying out the above-mentioned method are also disclosed.

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Almere, Netherlands

Bob Van Gemen