Rnase h-based assays utilizing modified RNA monomers

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...

AI Technical Summary

Benefits of technology

[0185]An important advantage of the present invention is that it allows double interrogation of the mutation site, and hence greater specificity, than standard ligation assays. There is an opportunity for discrimination of a variant allele both at the cleavage step and the ligation step.

[0186]Table 3 illustrates a non-comprehensive set of possible elements of the current invention to improve oligonucleotide ligation assays.TABLE 3Oligonucleotide Ligation AssayDonorAcceptorOligonucleotideOligonucleotideLabelingRNase HFlanking sequenceBlocking GroupBlocking GroupGroupCleavage SitemodificationsDivalentcationDNA LigaseNoneNoneNoneRNANoneMg2+Hot Start(5′-phosphate)1. Single1. Ab5′-OHModification ofFluorophoreRNA residueNuclease-resistant2. Chemically(Functional3′-terminal2. Multiplelinkagesmodifiedblock)residueRNA residues1. Phosphorothioate1. C3 spacer2. DithioateModification ofFluorophore / 3. Methyl-5′-residueQuencherphosphonate1. C3 spacer4. Non-nucleotide spacersDownstreamUpstreamEnzymeModifiedAlternativeNon-Hot Startmodificationmodification1. Horse-residues:divalentcation + / −1. Adjacent to the1. Adjacent to theradish1. 2 adjacent 2′-Mg2+5′-terminal3′-terminalperoxidaseF residuesresidueresidue2. Alkaline2. Further2. FurtherphosphatasedownstreamUpstreamBiotin2′OMeHapten1. DeoxigeninAntibodySecondaryMass TagmismatchesRadiolabel32P, 14C, 3H,35S, etc.DonorAcceptorOligonucleotideOligonucleotideReactionBlocking GroupBlocking GroupRNase HSampleUseConditionsAssay FormatNoneNoneRNase H1GenomicQuantificationRNase H cleavageStand alone(5′-phosphate)DNAof targetand DNA ligation1. Single cycle5′-OHModification ofRNase H2nucleic acidat single2. Linear(Functional3′-terminal1. Non-thermostablesequencetemperatureAmplificationblock)residue2. Thermostable1. Chromosomal3. LCRModification of1. C3 spacerA. Hot StartMitochondrial / copy number5′-residuei. Intrinsicchloroplast2. mRNA1. C3 spacerii. AbDNAiii. ChemicallymodifiedB. Non-Hot StartDownstreamUpstreamRNase H3 and otherDetection ofRNase H cleavageCoupled tomodificationmodificationcatalysts that cleavevariantof elevatedprimer extension1. Adjacent to the1. Adjacent to theRNA / DNAalleletemperature1. PCR5′-terminal3′-terminalheteroduplexes(reduced2. Reverseresidueresiduetemperature fortranscription2. Further2. FurtherDNA ligation)3. PolyampdownstreamUpstreamRNase H mutantscDNAhaving alteredcleavage specificity1. Enhanced cleavageof 2′-F substratesSequencing Reactions

[0187]In one embodiment, a method of sequencing a target DNA of interest is provided. The method entails

[0188](a) providing a reaction mixture comprising a primer having a cleavage domain and a blocking group linked at or near to the 3′ end of the primer which prevents primer extension, a sample nucleic acid comprising the target DNA sequence of interest, a cleaving enzyme, nucleotide triphosphate chain terminators (e.g., 3′ dideoxynucleotide triphosphates) and a polymerase,

[0189](b) hybridizing the primer to the target nucleic acid to form a double-stranded substrate;

[0190](c) cleaving the hybridized primer with the cleaving enzyme at a point within or adjacent to the cleavage domain to remove the blocking group from the primer; and

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.

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