Nucleobase analogs for degenerate hybridization devised through conformational pairing analysis.
A conformational pairing analysis was used to devise nucleobase analogs capable of forming nonselective and energetically favorable base pairs opposite either the purine or the pyrimidine constituents of nucleic acids. 5-methylisocytosine and isoguanine were conceived as a degenerate pyrimidine and a degenerate purine, respectively. Data from previous DNA duplex melting experiments verified that the analogs can act as degenerate nucleobases as hypothesized. Isoguanine also formed unusually stable base pairs with guanine. A quantitative PCR assay yielding equivalent results across hepatitis C virus (HCV) subtypes was created with this system, despite the use of a single probe targeted to a polymorphic region. Amplification curves using probes with 5-methylisocytosine or isoguanine opposite appropriate ambiguous target positions exhibited more signal than curves from similar probes containing common degenerate nucleobase hypoxanthine.
Abraham
Michelle L
ML
Siemens Medical Solutions Diagnostics, Berkeley, CA 94702, USA.
Albalos
Maria
M
Guettouche
Toumy
T
Friesenhahn
Michel J
MJ
Battersby
Thomas R
TR
D020029:Base Pairing; D015342:DNA Probes; D004279:DNA, Viral / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D016174:Hepacivirus / Q000235:genetics / Q000302:isolation & purification; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009692:Nucleic Acid Heteroduplexes; D009693:Nucleic Acid Hybridization / Q000379:methods*; D009711:Nucleotides / Q000737:chemistry*; D009841:Oligonucleotides / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction
D015342:DNA Probes; D004279:DNA, Viral; D009692:Nucleic Acid Heteroduplexes; D009711:Nucleotides; D009841:Oligonucleotides