Rnase h-based assays utilizing modified RNA monomers

a technology of rnase and assays, which is applied in the direction of biochemistry, organic chemistry, biochemical apparatus and processes, etc., can solve the problems of non-specific amplification, false positives in certain assays, and the reaction mixture cannot support primer extension at lower temperatures, so as to achieve greatly enhanced specificity

Inactive Publication Date: 2009-12-31
INTEGRATED DNA TECHNOLOGIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0026]The current invention provides novel biological assays that employ RNase H cleavage in relation to nucleic acid amplification, detection, ligation, sequencing, and synthesis. Additionally, the invention provides new assay formats to utilize cleavage by RNase H and novel oligonucleotide substrates for such assays. The compounds, kits, and methods of the present invention provide a convenient and economic means of achieving highly specific primer-based amplification reactions that are substantially free of nonspecific amplification impurities such as primer dimers. The methods and kits of the present invention avoid the need for reversibly inactivated DNA polymerase and DNA ligase enzymes.
[0027]One objective of the present invention is to enable hot start protocols in nucleic acid amplification and detection assays including but not limited to PCR, OLA (oligonucleotide ligation assays), LCR (ligation chain reaction), polynomial amplification and DNA sequencing, wherein the hot start component is a thermostable RNase H or other nicking enzyme that gains activity at the elevated temperatures employed in the reaction. Such assays employ a modified oligonucleotide of the invention that is unable to participate in the reaction until it hybridizes to a complementary nucleic acid sequence and is cleaved to generate a functional 5′- or 3′-end. Compared to the corresponding assays in which standard unmodified DNA oligonucleotides are used the specificity is greatly enhanced. Moreover the requirement for reversibly inactivated DNA polymerases or DNA ligases is eliminated.

Problems solved by technology

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.
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.
Interestingly, 2′-modification of the substrate duplex alters the geometry of the helix and can adversely affect activity of RNase H1.
Even a 2′-F nucleoside, which is the most “conservative” RNA analog with respect to changing chemical structure, adversely affects activity.
Despite improvements offered by these assays, there remain considerable limitations.
The PCR assays utilize a hot-start DNA polymerase which adds substantially to the cost.
In addition, the utility of these various assays has been limited by undesirable cleavage of the oligonucleotide probe or primer used in the reaction, including water and divalent metal ion catalyzed hydrolysis 3′ to RNA residues, hydrolysis by single-stranded ribonucleases and atypical cleavage reactions catalyzed by Type II RNase H enzymes at positions other than the 5′-phosphate of an RNA residue.

Method used

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  • Rnase h-based assays utilizing modified RNA monomers
  • Rnase h-based assays utilizing modified RNA monomers
  • Rnase h-based assays utilizing modified RNA monomers

Examples

Experimental program
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Effect test

example 1

Cloning of Codon Optimized RNase H2 Enzymes from Thermophilic Organisms

[0217]This example describes the cloning of codon optimized RNase H2 enzymes from thermophilic organisms.

[0218]To search for functional novel RNase H2 enzymes with potentially new and useful activities, candidate genes were identified from public nucleotide sequence repositories from Archaeal hyperthermophilic organisms whose genome sequences had previously been determined. While RNase H2 enzymes do share some amino acid homology and have several highly conserved residues present, the actual homology between the identified candidate genes was low and it was uncertain if these represented functional RNase H2 enzymes or were genes of unknown function or were non-functional RNase H2 genes. As shown in Table 4, five genes were selected for study, including two organisms for which the RNase H2 genes have not been characterized and three organisms to use as positive controls where the RNase H2 genes (rnhb) and function...

example 2

Expression of Recombinant RNase H2 Peptides

[0222]The following example demonstrates the expression of recombinant RNase H2 peptides.

[0223]The five synthetic gene sequences from Example 1 were subcloned using unique Bam HI and Hind III restriction sites into the bacterial expression vector pET-27b(+) (Novagen, EMD Biosciences, La Jolla, Calif.). This vector places six histidine residues (which together comprise a “His-tag”) (SEQ ID NO: 292) at the carboxy terminus of the expressed peptide (followed by a stop codon). A “His-tag” permits use of rapid, simple purification of recombinant proteins using Ni affinity chromatography, methods which are well known to those with skill in the art. Alternatively, the synthetic genes could be expressed in native form without the His-tag and purified using size exclusion chromatography, anion-exchange chromatography, or other such methods, which are also well known to a person of ordinary skill in the art.

[0224]BL21(DE3) competent cells (Novagen) w...

example 3

RNase H2 Activity for the Recombinant Peptides

[0230]The following example demonstrates RNase H2 activity for the recombinant peptides.

[0231]RNase H enzymes cleave RNA residues in an RNA / DNA heteroduplex. All RNase H enzymes can cleave substrates of this kind when at least 4 sequential RNA residues are present. RNase H1 enzymes rapidly lose activity as the RNA “window” of a chimeric RNA / DNA species is shortened to less than 4 residues. RNase H2 enzymes, on the other hand, are capable of cleaving an RNA / DNA heteroduplex containing only a single RNA residue. In all cases, the cleavage products contain a 3′-hydroxyl and a 5′-phosphate (see FIG. 1). When multiple RNA residues are present, cleavage occurs between RNA bases, cleaving an RNA-phosphate linkage. When only a single RNA residue is present, cleavage occurs only with Type II RNase H enzymes. In this case cleavage occurs on the 5′-side of the RNA base at a DNA-phosphate linkage (see FIG. 3). RNase H enzymes require the presence of...

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Abstract

The present invention pertains to novel oligonucleotide compounds for use in various biological assays, such as nucleic acid amplification, ligation and sequencing reactions. The novel oligonucleotides comprise a ribonucleic acid domain and a blocking group at or near the 3′ end of the oligonucleotide. These compounds offer an added level of specificity previously unseen. Methods for performing nucleic acid amplification, ligation and sequencing are also provided. Additionally, kits containing the oligonucleotides are also disclosed herein.

Description

[0001]Priority is claimed to U.S. provisional application No. 61 / 049,204 filed on Apr. 30, 2008, which is incorporated by reference in its entirety.FIELD OF THE INVENTION[0002]This invention pertains to methods of cleaving a nucleic acid strand to initiate, assist, monitor or perform biological assays.BACKGROUND OF THE INVENTION[0003]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 lower temperature conditions, the primers may bind non-specifically to other partia...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C12P19/34C07H21/02C07H21/04
CPCC12Q1/6844C12Q1/6853C12N9/96C12Q2549/101C12Q2525/186C12Q2521/301C12Q2525/121
Inventor WALDER, JOSEPH ALANBEHLKE, MARK AARONROSE, SCOTTDOBOSY, JOSEPH
Owner INTEGRATED DNA TECHNOLOGIES
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