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]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...

AI Technical Summary

Benefits of technology

[0182]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.

[0183]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 SitemodsDivalentcationDNA LigaseRNase HNoneNoneNoneRNANoneMg2+Hot StartRNase H1(5′-phosphate)1. Single1.Ab5′-OHModification ofFluorophoreRNA residuesNuclease-resistant2.Chem-RNase H2(Functional block)3′-terminal2. Multiple linkagesically1. Non-thermostableModification of residueFluorophore / RNA residues1. Phos- modifed2. Thermostable5′-residue1. C3 spacerQuencherphorothioateA. Hot Start1. C3 spacer2. Dithioatei. Intrinsic3. Methyl-ii. Abphosphonateiii. Chemically4. Non-nucleotidemodifiedspacersB. Non-Hot StartDownstreamUpstreamEnzymeModifiedAlternativeNon-HotRNase H3 and othermodificationmodification1. Horse-radishresidues:divalentStartcatalysts that cleave1. Adjacent to1. Adjacent to peroxidase1. 2 adjacent 2′cation + / −RNA / DNAthe 5′-terminalthe 3′-terminal2. AlkalineF residuesMg2+heteroduplexesresidueresiduephosphatase2. Further2. FurtherBiotin2′ OMeRNase H mutantsdownstreamupstreamHaptenhaving altered1. Deoxigenincleavage specificityAntibodySecondary1. Enhanced cleavage Mass Tagmismatchesof 2′-F substratesRadiolabel32P, 14C, 3H,35S, etc.ReactionSampleUseConditionsAssay FormatGenomicQuanitifcationRNase H cleavageStand-aloneDNAof targetand DNA ligation1. Single cyclenucleic acidat single2. LinearsequencetemperatureAmplificationMito-chondrial / 1. Chromosomal3. LCRchloropastcopy numberDNA2. mRNADetection ofRNase H cleavageCoupled tovariantat elevatedprimer alleletemperatureextension(reduced 1. PCRtemperature for2. ReversecDNADNA ligation)transcription3. PolyampSequencing Reactions

[0184]In one embodiment, a method of sequencing a target DNA of interest is provided. The method entails(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,(b) hybridizing the primer to the target nucleic acid to form a double-stranded substrate;(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(d) extending the primer with the polymerase.

[0185]In one embodiment, the invention is used in a “next generation” sequencing platform. One type of next generation sequencing is “sequencing by synthesis”, wherein genomic DNA is sheared and ligated with adapter oligonucleotides or amplified by gene-specific primers, which then are hybridized to complementary oligonucleotides that are either coated onto a glass slide or are placed in emulsion for PCR. The subsequent sequencing reaction either incorporates dye-labeled nucleotide triphosphates or is detected by chemiluminescence resulting from the reaction of pyrophosphate released in the extension reaction with ATP sulfurylase to generate ATP and then the ATP-catalyzed reaction of luciferase and its substrate luciferin to generate oxyluciferin and light.

[0186]A second type of next generation sequencing is “sequencing by ligation”, wherein four sets of oligonucleotides are used, representing each of the four bases. In each set, a fluorophore-labeled oligonucleotide of around 7 to 11 bases is employed in which one base is specified and the remaining are either universal or degenerate bases. If, for example, an 8-base oligonucleotide is used containing 3 universal bases such as inosine and 4 degenerate positions, there would be 44 or 256 different oligonucleotides in each set each with a specified base (A, T, C or G) at one position and a fluorescent label attached to either the 5′- or 3′-end of the molecule or at an internal position that does not interfere with ligation. Four different labels are employed, each specific to one of the four bases. A mixture of these four sets of oligonucleotides is allowed to hybridize to the amplified sample DNA. In the presence of DNA ligase the oligonucleotide hybridized to the target becomes ligated to an acceptor DNA molecule. Detection of the attached label allows the determination of the corresponding base in the sample DNA at the position complementary to the base specified within the oligonucleotide.

[0187]In one embodiment of the present invention, a donor oligonucleotide of about 7-11 bases contains a specified base at the 5′ end of the oligonucleotide. The remaining bases are degenerate or universal bases, and a label specific to the specified base is incorporated on the 3′ side of the specified base. The 3′ end of the probe is irreversibly blocked to prevent the donor oligonucleotide from also acting as an acceptor. In some cases this may be accomplished by the labeling group. The second base from the 5′ end of the oligonucleotide, i.e., the residue next to the specified base is a degenerate mixture of the 4 RNA bases. Alternatively, any anaolog recognized by RNase H2, such as a 2′-fluoronucleoside may be substituted at this position. A universal base such as riboinosine or ribo-5-nitroindole, may also be incorporated at this location. The probe first hybridizes to the target sequence and becomes ligated to the acceptor DNA fragment as in the standard sequencing by ligation reaction. After detection of the specified base, RNase H2 is added which cleaves the probe on the 5′-side of the RNA residue leaving the specified base attached to the 3′ end of the acceptor fragment. The end result is that the acceptor fragment is elongated by one base and now is in position to permit the determination of the next base within the sequence. The cycle is repeated over and over, in each case moving the position of hybridization of the donor oligonucleotide one base 3′ down the target sequence. The specificity is increased compared to traditional sequencing by ligation because the specified base is always positioned at the junction of the ligation reaction.

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.

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