Qiagen Gmbh

Nan Fang

Andreas Missel

Dirk Loeffert

Katja Heitz

Dirk Löffert

Lars-Erik Peters

Holger Wedler

Bernhard Noll

Isabell Czolkos

Christine Runde

Wolfgang Krebs

Christine Beckmann

The invention refers to a novel method of preparing strand-specific RNA-sequencing libraries that can be used to identify DNA coding and non-coding strands that are transcribed to RNA. Such strand-specific RNA-sequencing libraries are especially useful in discovering anti-sense RNA and non-coding RNA. Random primer oligonucleotides, covalently coupled to a moiety, which blocks ligation, are used for RT reaction or the subsequent generation of the second DNA strand so that only one strand of the generated double-stranded DNA is ligated to sequencing adapters at the 5' nucleotide and sequenced by paired-end sequencing.

Method for generating a RNA-sequencing library

The invention relates to a method for preparing a strand-specific library from an nucleic acid or preferably RNA sample, for RNA comprising the steps of: (i) optionally fragmenting said RNA sample, (ii) generating a plurality of first cDNA strands by subjecting said fragmented RNA to reverse transcription by using a reverse transcriptase and first oligonucleotide primers, (iii) generating a plurality of second cDNA strands by using a DNA polymerase, second oligonucleotide primers, and the plurality of first cDNA strands, and (iv) ligating adapters to the 3' and 5&#x2032; termini of the of double-stranded cDNA, (v) wherein the first cDNA strand allows no adapter ligation at its 5&#x2032; terminus and said second cDNA strand allows adapter ligation at its 5&#x2032; terminus, or vice versa, and, (v) optionally cloning, sequencing or otherwise using the strand-specific library. The invention also relates to a kit for preparing a strand-specific library from an RNA sample, wherein said kit comprises, (i) random oligonucleotide primers comprising a 5&#x2032; terminus nucleotide which allows no adaptor ligation, (ii) random oligonucleotide primers comprising a 5&#x2032; terminus nucleotide which allows adaptor ligation, (iii) optionally a reverse transcriptase for synthesizing a first cDNA strand complementary to the template RNA, (iv) optionally dNTPs, and (v) optionally a DNA polymerase.

Method for generating a stranded RNA library

The present invention is directed to methods for the generation of nucleic acids from an RNA template and further nucleic acid replication. Specifically, the invention is directed to the generation and amplification of nucleic acids by reverse transcriptase-polymerase chain reaction (RT-PCR).

Methods for RT-PCR comprising an anionic polymer

The present invention is directed to novel methods and kits to be employed for preparing adapter-ligated amplicons or a sequencing library of a target DNA, respectively.

Preparation of adapter-ligated amplicons

The present invention relates to methods and uses for the detection or quantification of newly-synthesized double-stranded target nucleic acid molecules in a sample during quantitative real-time polymerase chain reaction (qPCR) amplification. According to the invention, an intercalating dye recognizing double-stranded DNA molecules with higher affinity than single-stranded DNA molecules and a fluorophore-labeled oligonucleotide-probe being sequence specific for a target nucleic acid molecule are simultaneously employed, thus enabling quantification a specific target and total amount of a mixed nucleic acid population, and enabling assessing the cause of suboptimal PCR performance.

Nucleic acid detection and quantification

The present invention relates to methods and uses where a combination of (i) an enzyme exhibiting 5'&#x2192;3&#x2032; exonuclease activity and specifically degrading 5&#x2032;-phosphorylated strands of double-stranded nucleic acids is used, and (ii) one or more primer pair(s), each primer being 5&#x2032; phosphorylated, in a method comprising at least a first and a second amplification reaction of a first and a second template, respectively, for preventing contamination of the second amplification reaction with amplification products of the first amplification reaction, as well as novel methods and kits using the combination. Also, the present invention relates to kits comprising the enzyme and either 5&#x2032;-phosphorylated primers or a polynucleotide kinase.

Optimization of DNA amplification reactions

The present invention relates to a method for real-time monitoring and/or quantification of newly-synthesized complementary deoxyribonucleic acid (cDNA) during a reverse transcription reaction of an ribonucleic acid (RNA) template in a sample, the method using a fluorogenic dye binding to RNA:cDNA hybrids. The present invention also relates to the use of this method as well as to kits employing the fluorogenic dye.

Quantification of RNA

The present invention is in the fields of molecular biology. The present invention is directed to novel compositions, methods and kits useful for the generation of nucleic acids from an RNA template and further nucleic acid replication. Specifically, the invention is directed to the generation and amplification of nucleic acids by reverse transcriptase-polymerase chain reaction.

Composition and methods for RT-PCR comprising an anionic polymer

The invention relates to a method for the quantification of one or more nucleic acids in a sample, for example: making a sample available which contains at least one nucleic acid to be quantified, adding an oligonucleotide probe, the oligonucleotide probe comprising a sequence which can specifically hybridize to the nucleic acid to be quantified or to a common sequence of the nucleic acids to be quantified, incubating the sample under conditions which allow the hybridization of the oligonucleotide probe to the nucleic acid(s) to be quantified, incubating the sample under conditions which allow the extension of hybridized probes, the nucleic acid(s) serving as a template in each case, removing the non-hybridized probes from the sample and quantifying the hybridized oligonucleotide probes to measure the quantity of the nucleic acid(s) to be quantified. The invention also relates to a kit for carrying out said method.

Quantification of nucleic acids

The present invention relates to a method for detecting and/or for quantifying poly(A) RNA and/or mRNA, wherein a poly(dT) oligonucleotide which features a fluorescent dye and also a quencher is hybridized to the nucleic acid to be detected. Non-hybridized poly(dT) oligonucleotide is degraded by an added nuclease, and as a result, fluorescently labelled nucleotides are released and the resulting fluorescent signal is measured.

Method for detecting and quantifying poly(A) RNA and mRNA

The present invention relates to a thermophilic polymerase, wherein the DNA polymerase has an in-vitro primer extension rate that is &#x3e;35 bases/second and faster relative to the primer extension rate of a DNA polymerase comprising amino acid sequences SEQ ID NO: 2 or 4, when measured under identical conditions in a DNA replication assay using primed single strand M13mp18 DNA and an incubation temperature of 60&#xb0; C. The invention also relates to a vector comprising the polymerase, a host cell comprising the vector. The invention relates to a nucleic acid replication kit comprising the polymerase according to the invention.

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Hilden, Germany

Nan Fang