Qiagen Gmbh

Ralf Peist

Dirk Löffert

Mario Scherer

Miroslav Vranes

Stefan Otto Cornelius

Christian Korfhage

Holger Engel

Patrick Baumhof

Ralf Himmelreich

Christoph Erbacher

Alexander Azzawi

Roland Fabis

Dirk Loeffert

Andreas Missel

Jörg Dennig

Ingerlise Evans Haaland

Hege Hardersen

Margaretha König

Nicolas Rudinger

Thorsten Träger

Axel Deeg

Silvia Magyar

An oligonucleotide suitable for use as a detection probe in a method for evaluating the amplification efficiency, the presence of inhibitors, degradation and/or for performing a quantification analysis of a target nucleic acid in a real-time amplification reaction. The oligonucleotide probe is preferably SEQ ID NO. 4, a reverse complement of SEQ ID NO. 4, an oligonucleotide that shares 95% sequence identity with SEQ ID NO. 4, or an oligonucleotide that shares 95% sequence identity with a reverse complement of SEQ ID NO. 4. Also a kit including an internal nucleic acid control template, a set of primers for amplifying the internal nucleic acid control template, and the oligonucleotide probe.

Detection probe for internal amplification control and kit including same

A kit for use in assessing the status of nucleic acid degradation and/or the integrity of one or more nucleic acids in a sample by amplifying at least two overlapping regions within at least one locus and detecting the amount of the at least two amplification products. The kit includes a primer and at least two probes that bind under stringent conditions to a sequence that shares at least 80% sequence identity to a sequence selected from the group of sequences consisting of SEQ ID NO. 6 to SEQ ID NO. 47 over a stretch of 80 base pairs, or to a reverse complement thereof. One of the at least two probes binds to one of the at least two overlapping regions and the other of the at least two probes binds to a non-overlapping region.

Kit for determining nucleic acid degradation

The invention is in the field of regulation of enzymatic activity in nucleic acid modifying reactions. It describes a method of regulating enzymatic activity by adding chelating agents to the reaction composition and exploits the fact that both the binding of divalent cations to these chelating agents and the pH of commonly used buffers is temperature dependent. PCR experiments that are hampered by non-specific side products can be regulated such that the target sequence is amplified in a more specific manner.

Cation chelator hot start

PCR that allows the researchers to amplify a desired DNA requiring only tiny amounts of sample. Such amplification reactions are technically challenging and are often hampered by several practical issues such as the presence of PCR inhibitors, sample degradation and low quantities of said sample. The invention addresses these issues with a method for evaluating the amplification efficiency and/or the presence of inhibitors and/or degradation and/or performing a quantification of a nucleic acid in a real-time amplification reaction comprising: optionally amplifying in a reaction composition a first target nucleic acid using a first primer pair in a real-time amplification reaction, (i) amplifying in said reaction composition one or more second internal nucleic acid control templates (IC) with a length of between 50 and 2000 nucleotides, wherein the second nucleic acid has a sequence selected from the group of: i) SEQ ID NO. 1 or a sequence that is differs by no more than 15% therefrom, ii) a reverse complement of SEQ ID NO. 1 or a sequence that is differs by no more than 15% therefrom, iii) a sequence or a reverse complement thereof comprising the final 18 to 30 3'-nucleotides of SEQ ID NO. 1 at its terminal 3&#x2032;-end and the final 18 to 30 5&#x2032;-nucleotides of SEQ ID NO. 1 at its terminal 5&#x2032;-end, or terminal ends that differ by no more than 5% from SEQ ID NO. 1, wherein between these terminal 3&#x2032;-ends and 5&#x2032;-ends the nucleic may have any nucleotide sequence and is between about 30 and about 1950 nucleotides in length.

Internal amplification control

The present invention relates to a method for assessing the status of nucleic acid degradation and/or integrity of one or more nucleic acids in a sample, comprising the steps amplifying at least two overlapping regions within at least one locus (e.g. by heminested or nested PCR), and detecting the amount of the at least two amplification products through the use of at least two probes, wherein one probe binds to the region of overlap and the at least one other probe binds to a non-overlapping region. The invention further relates to a method of designing primers and/or probes for amplifying at least two overlapping regions within at least one locus, wherein the locus that is amplified is a single copy locus (SCL) or multicopy locus (MLC). The invention also relates to a primer and a primer pair for amplifying at least two overlapping regions from one nucleic acid template which is a multicopy locus present in 21 loci in the human genome. These primers and probes may be in a kit. Hence, the invention also relates to a kit for assessing the status of nucleic acid degradation and/or integrity of one or more nucleic acids in a sample.

Method for determining nucleic acid degradation in a sample in which at least two overlapping amplicons are produced and two probes are used in the method

According to a first aspect of the present invention, a method is provided for detecting and/or quantifying male genomic DNA in a sample, wherein the method comprises the step of amplification of a multicopy locus within the human Y-chromosome (MCL-Y), wherein said locus shares at least 80% sequence identity to a sequence according to SEQ ID NO. 3 over a stretch of at least 60 base pairs (bp). A second aspect of the present invention relates to a primer or primer pair which hybridizes under stringent conditions to a sequence according to SEQ ID NO. 3 and/or any of 4 to 11. The invention also relates to a kit.

Method for quantifying and/or detecting human male DNA

Caution chelator hot start

The present invention relates to a reaction mixture for the amplification of nucleic acids, the non-methylated cytosine bases of which have been converted to uracil bases by means of a bisulfition reaction. The invention also discloses methods for amplifying bisulfited nucleic acid and for determining the nucleic acid methylation state, and also kits based on the reaction mixture according to the invention.

Amplification of bisulfite-reacted nucleic acids

The present invention relates to an improved method for isolating nucleic acids, particularly genomic desoxyribonucleic acid (DNA) from blood.

Method for extracting nucleic acid from blood

The present invention concerns a method for a polymerase chain reaction, in which a template nucleic acid, at least one primer, deoxyribonucleoside triphosphates as well as a DNA polymerase with proofreading activity are used. In addition, according to this invention, at least one target substrate is added to the polymerase chain reaction, whereby the efficiency of the DNA polymerase with proofreading activity is significantly increased. Any molecule that reduces or, in the optimal case, blocks the 3',5&#x2032;-exonuclease activity of the DNA polymerase used is suitable as target substrate. Technical solutions for the added substrate (target substrate) are in particular single stranded, linear oligonucleotides, hairpin oligonucleotides and RNA and DNA molecules. Furthermore, a kit is disclosed which comprise the required reagents for the implementation of the method according to the invention.

Method for polymerase chain reactions with use of a DNA polymerase with proofreading properties

The present invention concerns a method for inserting one or more tag sequences into a nucleic acid characterized by the following steps: (a) preparation of a template nucleic acid; (b) hybridization of at least one anchor sequence of at least one anchor oligonucleotide with one sequence section of the template nucleic acid; and (c) synthesization of a new strand of nucleic acid, which is partially complementary to the template nucleic acid and which contains a sequence complementary to the non-hybridized portion of the anchor oligonucleotide, e.g. to at least one tag sequence, on its 3' end.

Insertion of sequence elements into nucleic acids

Disclosed are methods for the enhancement of the reactivation of thermostable reversibly inactivated enzymes comprising reactivating at least one thermostable reversibly inactivated enzyme in the presence of one or more nitrogen containing compounds.

Enhancing reactivation of thermostable reversibly inactivated enzymes

The invention concerns a method for the detection of cytosine methylations in DNA and a kit for undertaking an assay according to the method of the invention.

Process for the detection of cytosine methylations

The invention relates to a method for processing RNA, in particular, a RNA reaction method and kits for carrying out said RNA reaction method.

Growing community of inventors

Hilden, Germany

Ralf Peist