Introduction

In degenerate hybridization a single nucleic acid sequence hybridizes nonselectively to target sequences containing polymorphic sites. Degenerate probes are created from otherwise specific sequences with nucleobase analogs acting as degenerate bases opposite ambiguous target sites. Interaction of an analog and intended target nucleobases should be nonselective and should not be markedly destabilizing relative to Watson-Crick interaction. Degenerate probes offer the advantage of reducing the number and total amount of probes required to target ambiguous sites. Fewer probes simplify assay optimization, reduce background signal, and potentially reduce assay costs. A degenerate site need not be opposite a primary polymorphic site interrogated by a hybridization probe; it may simply allow targeting of regions with incidental sequence variation.

A significant challenge of designing a degenerate base is to compensate for the different hydrogen bonding patterns presented by the nucleobase constituents of nucleic acids (A, C, G, and T/U). Previous strategies for nonselective pairing with two or more of the nucleobases include tautomerization (1), bond rotation to present different hydrogen bonding faces (2,3), or by dispensing with hydrogen bonding altogether (4,5,6). Here we employ a systematic conformational pairing analysis of nucleobase analogs to devise degenerate bases. Using this approach, 5-methylisocytosine (F) and isoguanine (J) were conceived as degenerate nucleobases that minimally destabilize nucleic acid duplexes opposite purine (R) and pyrimidine (Y) ambiguous sites, respectively.

Materials and Methods

Oligodeoxyribonucleotides

A 27-mer forward and 27-mer reverse primer pair, targeted to sequence regions conserved across all targets examined, was used. Primers were obtained from Operon Biotechnologies (Huntsville, AL, USA) with reversed-phase high-performance liquid chromatography (HPLC) purification. Probes 1–8 were 5′-labeled with 6-fluorescein and 3′-labeled with Black Hole Quencher 1. Probes 1 and 2, 4–6, and 8 were obtained from Biosearch Technologies (Novato, CA, USA) with anion exchange and reversed-phase HPLC purification. Probes 3 and 7 containing F or J were synthesized on a 394 DNA/RNA Synthesizer (Applied Biosystems, Foster City, CA, USA). Phosphoramidites were purchased from Glen Research (Sterling, VA, USA). Solid support with Black Hole Quencher was purchased from Biosearch Technologies. The oligodeoxynucleotides (ODNs) were purified by reversed-phase HPLC using the Wave System (Transgenomic, Omaha, NE, USA). ODN mass was verified as being within 0.5% of the expected mass by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry using a Voyager-DE (Applied Biosystems). ODN purity was ≥93% by electrophoresis in a polyacrylamide-filled capillary on a ^3DCE instrument (Agilent Technologies, Santa Clara, CA, USA).

Probes against sense strand of PCR product were: probe 1, 5′-TTTCGCGACCCAACACTACTCGGCT-3′; probe 2, 5′-TTTCGCAACCCAACGCTACTCGGCT-3′; probe 3, 5′-TTTCGCJACCCAACJCTACTCGGCT-3′; probe 4, 5′-TTTCGCIACCCAACICTACTCGGCT-3′. Probes against antisense strand of PCR product were: probe 5, 5′-AGCCGAGTAGTGTTGGGTCGCGAAA-3′; probe 6, 5′-AGCCGAGTAGCGTTGGGTTGCGAAA-3′; probe 7, 5′-AGCCGAGTAGFGTTGGGTFGCGAAA-3′; probe 8, 5′-AGCCGAGTAGIGTTGGGTIGCGAAA-3′. Bold nucleobases are opposite target polymorphic sites.

Hepatitis C Virus Transcripts

Hepatitis C virus (HCV) transcripts were produced from viral samples of subtypes 1a, 1b, 2a, and 2b and quantified by phosphate analysis, as described previously (7).

Reverse Transcription PCR with HCV Transcripts

Reverse transcription PCR (RTPCR) was conducted in 96-well microplates in 25-µL volumes. Individual wells contained 1 µL OneStep RT-PCR enzyme mix (Qiagen, Valencia, CA, USA), 5.0 mM MgCl₂, 0.3 mM each deoxynucleoside triphosphate, and 30 nM ROX reference dye (Stratagene, La Jolla, CA, USA) in OneStep RT-PCR buffer. Each reaction contained 400 nM each primer and 125 nM probe. Target levels of 1 × 10³, 1 × 10⁵, and 1 × 10⁷ copies of HCV transcript and no template controls were run in triplicate. Amplification was performed on a Stratagene Mx3000P with 50°C reverse transcription incubation for 40min; 95°C Taq polymerase activation for 15 min; and 40 cycles of 95°C for 15 s; 67°C for 1 min; and 72°C for 30 s. MxPro software determined threshold cycles (C_T) employing amplification-based threshold (set at 5%), adaptive baseline, and moving average algorithms.