Promega Corporation

James Robert Hartnett

Trent Gu

Keith V Wood

John William Shultz

Martin Ken Lewis

Donna M Leippe

Michelle N Mandrekar

Christine Ann Andrews

Roy Welch

Michael R Slater

Fen Huang

Daniel Kephart

Richard Byron Rhodes

Ryan J Olson

Katharine M Miller

John E Monahan

James W Schumm

Leopoldo G Mendoza

Richard A Martinelli

Steven J Ekenberg

Mark A Maffitt

Randall L Dimond

Edward E Pahuski

Geoffrey R Hudson

Christopher L Jones

Ethan Edward Strauss

Mark W Knuth

Susanne Selman

Kimberly K Knoche

Paul Otto

Douglas R Storts

Alexander Novikov

Elena Bolchakova

Galina A Velikodvorskaya

The invention provides vectors and methods for directional cloning.

Vectors for directional cloning

The present invention relates to reverse transcription of RNA, and in particular to reverse transcription by thermostable DNA polymerases.andpossess reverse transcriptase activity in the presence of magnesium or manganese ions. Methods, compositions, and kits for reverse transcription and RT-PCR are also provided.

Method of reverse transcription

The present invention provides compositions comprising thermostable DNA polymerases derived from hyperthermophilic eubacteria. In particular, the present invention comprises thermostable DNA polymerases from the hyperthermophilic eubacterial species. The present invention also provides methods for utilizing naturally-occurring and non-naturally-occurring forms ofDNA polymerase in sequencing, reverse transcription, and amplification reactions.

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Processes are disclosed using the depolymerization of a nucleic acid hybrid to qualitatively and quantitatively analyze for the presence of a predetermined nucleic acid. Applications of those processes include the detection of single nucleotide polymorphisms, identification of single base changes, speciation, determination of viral load, genotyping, medical marker diagnostics, and the like.

Detection of nucleic acid hybrids

The present invention entails methods and kits for carrying them out based on the discovery that an RNA replicase, such as Q&bgr; replicase, has DNA-dependent RNA polymerase (&ldquo;DDRP&rdquo;) activity with nucleic acid segments, including DNA segments and DNA:RNA chimeric segments, which comprise a 2&prime;-deoxyribonucleotide or an analog thereof and which have sequences of RNAs that are autocatalytically replicatable by the replicase.

Nucleic acid amplification with DNA-dependent RNA polymerase activity of RNA replicases

Processes are disclosed using the depolymerization of a nucleic acid hybrid to qualitatively and quantitatively analyze for the presence of predetermined nucleic acid target sequences using a multiplex assay format. Applications of those processes include the detection of single nucleotide polymorphisms, identification of single base changes, speciation, genotyping, medical marker diagnostics, and the like.

Multiplex method for nucleic acid detection

The present invention provides compositions comprising thermostable DNA polymerases derived from hyperthermophilic eubacteria. In particular, the present invention comprises thermostable DNA polymerases from the hyperthermophilic eubacterial species . The present invention also provides methods for utilizing naturally-occurring and non-naturally-occurring forms of DNA polymerase in sequencing, reverse transcription, and amplification reactions.

Thermophilic DNA polymerases from Thermoactinomyces vulgaris

The present invention relates to reverse transcription of RNA, and in particular to reverse transcription by thermostable DNA polymerases. and possess reverse transcriptase activity in the presence of magnesium or manganese ions. Methods, compositions, and kits for reverse transcription and RT-PCR are also provided.

Processes are disclosed using the depolymerization of a nucleic acid hybrid to qualitatively and quantitatively analyze for the presence of a predetermined endogenous nucleic acid. Applications of those processes include the detection of single nucleotide polymorphisms, identification of single base changes, speciation, determination of viral load, genotyping, medical marker diagnostics, and the like.

The present invention entails methods, and kits for carrying them out, based on the discovery that an RNA replicase, such as Q&bgr; replicase, has DNA-dependent RNA polymerase (&ldquo;DDRP&rdquo;) activity with nucleic acid segments, including DNA segments and DNA:RNA chimeric segments, which comprise a 2&prime;-deoxyribonucleotide or an analog thereof and which have sequences of RNAs that are autocatalytically replicatable by the replicase. The discovery of this DDRP activity provides methods of the invention for nucleic acid amplification wherein a nucleic acid, with a DNA segment with the sequence of an RNA that is autocatalytically replicatable by an RNA replicase, is provided as a substrate for the replicase. The replicase catalyzes synthesis, from the DNA segment, of the RNA, which the replicase then autocatalytically replicates. The invention entails use of the amplification methods in detecting nucleic acid analytes, as in nucleic acid probe hybridization assays. Such assays of the invention include those wherein a nucleic acid analyte is hybridized with one or more nucleic acid probes, which include or are processed to generate a DNA segment which is amplifiable through production from the segment, catalyzed by the DDRP activity of an RNA replicase, of an autocatalytically replicatable RNA, which is autocatalytically replicated to provide an abundance of readily detectable reporter molecules. The invention permits replacement of an RNA, that is autocatalytically replicatable with an RNA replicase and employed as a reporter or label in prior art assays, such as nucleic acid probe hybridization assays or immunoassays, with a nucleic acid comprising a DNA segment with the same base sequence as the RNA. The invention also includes the methods of the invention with Mn , Co , or Zn in the solutions in which the DDRP activity occurs.

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Madison, WI, United States of America

James Robert Hartnett