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Rnase h-based assays utilizing modified RNA monomers

a technology of rnase and assays, which is applied in the field of rnase h-based assays utilizing modified rna monomers, can solve the problems of non-specific amplification giving rise in certain assays to false positives, significant decrease in the efficiency of amplification of the desired sequence, and reaction mixtures that cannot support primer extension at lower temperatures, etc., to achieve the effect of greatly enhancing specificity

Inactive Publication Date: 2015-08-13
INTEGRATED DNA TECHNOLOGIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides modified oligonucleotides that can be used in nucleic acid amplification and detection assays, such as PCR, without causing undesired reactions or interfering with the activity of DNA polymerases or ligases. These modifications include the use of thermostable RNase H or other nicking enzymes that gain activity at elevated temperatures. The modified oligonucleotides are unable to participate in the reaction until they hybridize to a complementary nucleic acid sequence and are cleaved by the enzyme. This greatly enhances the specificity of the assay compared to unmodified oligonucleotides. The position of the modification can be adjusted to alter specificity and the amount of enzyme needed for optimal cleavage rates. The invention also provides oligonucleotides that prevent the oligonucleotide from serving as a template for DNA synthesis and interfering with PCR. These modifications can be used in various nucleic acid amplification and detection assays without interfering with the assay's specificity.

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.
Furthermore, this approach may be inefficient because the large numbers of mononucleotides present in the reaction will absorb much of the UV light.
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 requirement for dUTP and the UNG enzyme adds significantly to the cost of performing PCR.
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
Comparison scheme
Effect test

example 1

Cloning of Codon Optimized RNase H2 Enzymes from Thermophilic Organisms

[0204]This example describes the cloning of codon optimized RNase H2 enzymes from thermophilic organisms.

[0205]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

[0214]The following example demonstrates the expression of recombinant RNase H2 peptides.

[0215]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.

[0216]BL21(DE3) competent cells (Novagen) w...

example 3

RNase H2 Activity for the Recombinant Peptides

[0222]The following example demonstrates RNase H2 activity for the recombinant peptides.

[0223]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]This application is a continuation application of U.S. application Ser. No. 12 / 507,142, filed Jul. 2, 2009 (now U.S. Pat. No. 8,911,948), which in turn is a continuation-in-part of U.S. application Ser. No. 12 / 433,896, filed Apr. 30, 2009, which in turn claims benefit under 35 U.S.C. 119(e) to U.S. provisional application No. 61 / 049,204 filed on Apr. 30, 2008. All of the above-identified applications are hereby incorporated by reference in their 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 exten...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68
CPCC12Q1/6848C12Q1/6853C12Q1/6858C12Q1/686C12Q1/6844C12Q2549/101C12Q2525/186C12Q2525/121C12Q2521/301
Inventor WALDERBEHLKE, MARK AARONROSE, SCOTTDOBOSY, JOSESPH
Owner INTEGRATED DNA TECHNOLOGIES
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