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Rnase-H-Based Assays Utilizing Modified Rna

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Rnase-H-Based Assays Utilizing Modified Rna (19) TZZ ¥_T (11) EP 2 279 263 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12Q 1/68 (2006.01) 04.09.2013 Bulletin 2013/36 (86) International application number: (21) Application number: 09739895.2 PCT/US2009/042454 (22) Date of filing: 30.04.2009 (87) International publication number: WO 2009/135093 (05.11.2009 Gazette 2009/45) (54) RNASE-H-BASED ASSAYS UTILIZING MODIFIED RNA MONOMERS TESTS AUF RNASE-H-BASIS MIT MODIFIZIERTEN RNA-MONOMEREN DOSAGES À BASE DE RNASE-H UTILISANT DES MONOMÈRES D’ARN MODIFIÉS (84) Designated Contracting States: • ROSE, Scott AT BE BG CH CY CZ DE DK EE ES FI FR GB GR Coralville, IA 52241 (US) HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL • DOBOSY, Joseph PT RO SE SI SK TR Coralville, IA 52241 (US) (30) Priority: 30.04.2008 US 49204 P (74) Representative: Grünecker, Kinkeldey, Stockmair & Schwanhäusser (43) Date of publication of application: Leopoldstrasse 4 02.02.2011 Bulletin 2011/05 80802 München (DE) (60) Divisional application: (56) References cited: 13173388.3 EP-A- 1 367 136 WO-A-01/21813 13173389.1 WO-A-03/074724 WO-A-2004/059012 13173390.9 WO-A-2007/062495 WO-A2-2005/021776 13173391.7 WO-A2-2007/141580 US-A- 5 744 308 US-A- 5 830 664 (73) Proprietor: Integrated Dna Technologies, Inc. Coralville, IA 52241 (US) • ITAYA M ET AL: "Molecular cloning of a ribonuclease H (RNase HI) gene from an extreme (72) Inventors: thermophile Thermus thermophilus HB8: a • WALDER, Joseph, Alan thermostable RNase H can functionally replace Chicago, IL 60645 (US) the Escherichia coli enzyme in vivo." NUCLEIC • BEHLKE, Mark, Aaron ACIDS RESEARCH 25 AUG 1991, vol. 19, no. 16, Coralville, IA 52241 (US) 25 August 1991 (1991-08-25), pages 4443-4449, XP002552896 ISSN: 0305-1048 Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 279 263 B1 Printed by Jouve, 75001 PARIS (FR) EP 2 279 263 B1 Description FIELD OF THE INVENTION 5 [0001] This invention pertains to methods of cleaving a nucleic acid strand to initiate, assist, monitor or perform biological assays. BACKGROUND OF THE INVENTION 10 [0002] The specificity of primer-based amplification reactions, such as the polymerase chain reaction (PCR), largely depends on the specificity of primer hybridization with a DNA template. Under the elevated temperatures used in a typical amplification reaction, the primers ideally hybridize only to the intended target sequence and form primer extension products to produce the complement of the target sequence. However, amplification reaction mixtures are typically assembled at room temperature, well below the temperature needed to insure primer hybridization specificity. Under 15 lower temperature conditions, the primers may bind non-specifically to other partially complementary nucleic acid se- quences or to other primers and initiate the synthesis of undesired extension products, which can be amplified along with the target sequence. Amplification of non- specific primer extension products can compete with amplification of the desired target sequences and can significantly decrease the efficiency of the amplification of the desired sequence. Non-specific amplification can also give rise in certain assays to a false positive result. 20 [0003] One frequently observed type of non-specific amplification product in PCR is a template independent artifact of the amplification reaction known as "primer dimers". Primer dimers are double-stranded fragments whose length typically is close to the sum of the two primer lengths and are amplified when one primer is extended over another primer. The resulting duplex forms an undesired template which, because of its short length, is amplified efficiently. [0004] Non- specific amplification can be reduced by reducing the formation of primer extension products ( e.g., primer 25 dimers) prior to the start of the reaction. In one method, referred to as a "hot- start" protocol, one or more critical reagent s are withheld from the reaction mixture until the temperature is raised sufficiently to provide the necessary hybridization specificity. In this manner, the reaction mixture cannot support primer extension at lower temperatures. Manual hot- start methods, in which the reaction tubes are opened after the initial high temperature incubation step and the missing reagents are added, are labor intensive and increase the risk of contamination of the reaction mixture. 30 [0005] Alternatively, a heat sensitive material, such as wax, can be used to separate or sequester reaction components, as described in U.S. Pat. No. 5, 411, 876, and Chou et al., 1992, Nucl. Acids Res. 20 (7) : 1717- 1723. In these methods, a high temperature pre- reaction incubation melts the heat sensitive material, thereby allowing the reagents to mix. [0006] Another method of reducing the formation of primer extension products prior to the start of PCR relies on the heat-reversible inactivation of the DNA polymerase. U.S. Pat. Nos. 5,773,258 and 5,677,152. describe DNA polymerases 35 reversibly inactivated by the covalent attachment of a modifier group. Incubation of the inactivated DNA polymerase at high temperature results in cleavage of the modifier- enzyme bond, thereby releasing an active form of the enzyme. Non- covalent reversible inhibition of a DNA polymerase by DNA polymerase-specific antibodies is described in U.S. Pat. Nos. 5,338,671. [0007] WO 2007/062495 discloses methods for adding a terminal sequence tag to nucleic acid molecules for use in 40 RNA or DNA amplification; the tag introduced provides means for identification and cloning of the 5’- end or the complete sequence of mRNAs. [0008] US 5,744,308 teaches a chimera oligonucleotide that can be used in a process for obtaining transcripts and/or amplification of a target sequence of a nucleic acid, having, at its 3’ end, a downstream sequence. [0009] EP 1367136 discloses a nucleotide useful for detecting a base substitution in a gene, a method for detecting 45 a base substitution in a gene using said nucleotide, and a kit for the same. [0010] WO 2005/021776 teaches methods for using mammalian RNase H, including human RNase H, and composi- tions thereof, particularly for reduction of a selected cellular RNA target via antisense technology. [0011] WO 2007/141580 discloses genes for components of RNase H2, namely RNASEH2A, RNASEH2B and RNASEH2C, together with the proteins encoded thereby. 50 [0012] One objective of the present invention can be used, for example, to address the problem of carry- over cross contamination which is a significant concern in amplification reactions, especially PCR wherein a large number of copies of the amplified product are produced. In the prior art, attempts have been made to solve this problem in a number of ways. For example, direct UV irradiation can effectively remove contaminating DNA (Rys & Persing, 1993, J Clin Microbiol. 31 (9) : 2356- 60 and Sarkar & Sommer, 1990 Nature. 343 (6253) : 27) but the irradiation of the PCR reagents must 55 take place before addition of polymerase, primers, and template DNA. Furthermore, this approach may be inefficient because the large numbers of mononucleotides present in the reaction will absorb much of the UV light. An alternative, the "UNG method", incorporates dUTP into the amplified fragments to alter the composition of the product so that it is different from native, naturally occurring DNA (Longo et al. 1990, Gene, 93 (1) : 125- 128) . The enzyme Uracil- N- 2 EP 2 279 263 B1 Glycosylase (UNG) is added together with the other components of the PCR mixture. The UNG enzyme will cleave the uracil base from DNA strands of contaminating amplicons before amplification, and render all such products unable to act as a template for new DNA synthesis without affecting the sample DNA. The UNG enzyme is then heat- inactivated and PCR is then carried out. The requirement for dUTP and the UNG enzyme adds significantly to the cost of performing 5 PCR. [0013] Another objective of the present invention is to provide PCR assays in which a hot-start reaction is achieved through a coupled reaction sequence with a thermostable RNase H. Ribonuclease Enzymes 10 [0014] Ribonucleases (RNases) are enzymes that catalyze the hydrolysis of RNA into smaller components. The enzymes are present internally; in bodily fluids; on the surface of skin; and on the surface of many objects, including untreated laboratory glasswear. Double- stranded RNases are present in nearly all intracellular environments and cleave RNA-containing, double-stranded constructs. Single- stranded RNases are ubiquitous in extracellular environments, and 15 are therefore extremely stable in order to function under a wide range of conditions. [0015] The RNases H are a conserved family of ribonucleases which are present in all organisms examined to date. There are two primary classes of RNase H: RNase H1 and RNase H2. Retroviral RNase H enzymes are similar to the prokaryotic RNase H1. All of these enzymes share the characteristic that they are able to cleave the RNA component of an RNA:DNA heteroduplex. The human and mouse RNase H1 genes are 78% identical at the amino acid level 20 (Cerritelli, et al., (1998) Genomics, 53, 300-307). In prokaryotes, the genes are named rnha (RNase H1) and rnhb (RNase H2). A third family of prokaryotic RNases has been proposed, rnhc (RNase H3) (Ohtani, et al. (1999) Biochemistry, 38, 605-618). [0016] Evolutionarily, "ancient" organisms (archaeal species) in some cases appear to have only a single RNase H enzyme which is most closely related to the modem RNase H2 enzymes (prokaryotic) (Ohtani, et al., J Biosci Bioeng, 25 88, 12- 19) .
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