Oligonucleotides containing pyrazolo[3,4-d] pyrimidines for hybridization and mismatch discrimination

Inactive Publication Date: 2010-09-09
ELITECH HLDG
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  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0008]When the guanine bases in an oligonucleotide are replaced by the guanine analogue ppG, the Tm values of probes containing the analogues are slightly higher than those of oligonucleotide probes containing guanine. Hence, G-containing and ppG-containing oligonucleotides perform similarly in hybridization assays. However, when ppG-substituted oligonucleotides are used as hydrolyzable probes (described infra and see U.S. Pat. No. 5,210,015), two properties are significantly enhanced. First, ppG-substituted probes are more effective at mismatch discrimination, as measured by higher signal-to-noise values comparing the fluorescent signal obtained from a perfectly-matched hybrid with that from a hybrid containing a single-nucleotide mismatch. In addition, ppG-substituted probes provide higher absolute signal from a perfectly-matched target.

Problems solved by technology

Thus, the potential discriminatory power of short oligonucleotides cannot be easily realized except under conditions of low stringency, which counteract their discriminatory ability.

Method used

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  • Oligonucleotides containing pyrazolo[3,4-d] pyrimidines for hybridization and mismatch discrimination
  • Oligonucleotides containing pyrazolo[3,4-d] pyrimidines for hybridization and mismatch discrimination
  • Oligonucleotides containing pyrazolo[3,4-d] pyrimidines for hybridization and mismatch discrimination

Examples

Experimental program
Comparison scheme
Effect test

example 1

Preparation of Dual-Labeled, MGB-Conjugated Hydrolyzable Probes

[0077]Synthesis of Oligonucleotide Probes Carrying a 5′-Reporting Dye

[0078][(3′,6′-dipivaloylfluoresceinyl)-6-carboxamidohexyl]-1-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite (6-FAM) and 3′-CDPI3-tail (Scheme 1). Oligonucleotides with a conjugated CDPI3 tail were prepared on a 1 μmol scale using standard 3′-phosphoramidite chemistry on a CDPI3-CPG support (˜20-50 mg) Preparation of the CDPI3-CPG support is disclosed in Lukhtanov et al. (1995) Bioconj. Chem. 6:418-426. Oligonucleotides lacking a conjugated MGB were synthesized by standard procedures. Synthesis was performed on an ABI 394 according to the protocol supplied by the manufacturer with one exception: 0.01 M (instead of the standard 0.1 M) iodine solution was utilized in the oxidation step to avoid iodination of the CDPI3 moiety, when CDPI3-conjugated oligonucleotides were being synthesized. An amino-linker for postsynthetic incorporation of the TAMRA dye ...

example 2

Target, Primer and Probe Sequences

[0082]Strategy

[0083]The target sequence is located in the E. coli supF gene contained in the plasmid pSP189 (FIG. 1, SEQ ID NO.: 1). See Parris et al. (1992) Gene 117:1-5. Binding sites for the primers used for amplification are indicated as Primer 1 and Primer 2, with Primer 1 having a sequence and polarity that is identical to that shown in FIG. 1, and Primer 2 having a sequence and polarity that is the reverse complement to that shown in FIG. 1. Three probes having overlapping sequences, each labeled with FAM at the 5′-end and with the quencher TAMRA at the 3′-end with were synthesized: a 12-mer, a 15-mer and an 18-mer. The 12-mer and 15-mer additionally contained a conjugated minor groove binder (CDPI3) near the 3′-end of the oligonucleotide. Finally, each probe contained either normal guanine residues (indicated by G in the Tables) or all of its guanine residues were substituted with ppG (indicated by ppG in the Tables). These probes were used ...

example 3

Hydrolyzable Probe Assay

[0086]Hydrolyzable probe assays with fluorescent monitoring were performed in an Idaho Technologies Light Cycler. Wittwer et al. (1997a) BioTechniques 22:130-138, and Wittwer et al. (1997b) BioTechniques 22:176-181. Each reaction mixture contained:[0087]40 mM NaCl[0088]20 mM Tris-Cl, pH 8.9[0089]5 mM MgSO4 [0090]0.05% (w / v) Bovine Serum Albumin[0091]125 μM each dATP, dGTP, dCTP, dTTP[0092]0.5 μM each primer[0093]0.5 μM probe[0094]0.5 U / 10 μL Taq Polymerase

[0095]Cycling conditions were 40 cycles of 0 sec at 94° C. (i.e., temperature was raised to 94° C. and immediately lowered to the annealing / extension temperature), then 15 sec at the annealing / extension temperature (which varied from 55-75° C. in individual experiments; see below and in figure legends for details). Fluorescent output was expressed as the ratio of fluorescence at 515-560 nm (fluorescein) to that at 560-630 nm (rhodamine), as analyzed by the manufacturer's software that was provided with the l...

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Abstract

Oligonucleotides in which one or more purine residues are substituted by pyrazolo[3,4-d]pyrimidines exhibit improved hybridization properties. Oligonucleotides containing pyrazolo[3,4-d]pyrimidine base analogues have higher melting temperatures than unsubstituted oligonucleotides of identical sequence. Thus, in assays involving hybridization of an oligonucleotide probe to a target polynucleotide sequence, higher signals are obtained. In addition, mismatch discrimination is enhanced when pyrazolo[3,4-d]pyrimidine-containing oligonucleotides are used as hybridization probes, making them useful as probes and primers for hybridization, amplification and sequencing procedures, particularly those in which single- or multiple-nucleotide mismatch discrimination is required.

Description

TECHNICAL FIELD[0001]This application is in the field of molecular biology relating to the use of oligonucleotides as probes and primers. It relates further to the use of modified nucleic acid bases to improve the hybridization properties and discriminatory abilities of oligonucleotides that are used as probes and primers.BACKGROUND[0002]Many techniques currently in use in molecular biology utilize oligonucleotides as probes and / or primers. It is often advantageous, in the practice of these techniques, to be able to distinguish between two or more sequences which are related but which differ by one or more nucleotides. For example, many mutations of clinical significance differ by only a single nucleotide from the wild-type sequence. Polymorphisms in mammalian genomes are also often characterized by sequence differences of one or a few nucleotides. The ability to make such a distinction is known as mismatch discrimination. In practical terms, mismatch discrimination describes the pr...

Claims

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Application Information

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IPC IPC(8): C07H21/00C12Q1/68C12N15/09C12Q1/6818C12Q1/6827C12Q1/6832
CPCC12Q1/6818C12Q1/6827C12Q1/6832C12Q2527/107C12Q2525/117
Inventor MEYER, JR., RICH B.AFONINA, IRINA A.KUTYAVIN, IGOR V.
Owner ELITECH HLDG
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