title: Hybridization properties of long nucleic acid probes for detection of variable target sequences, and development of a hybridization prediction algorithm.
abstract: One of the main problems in nucleic acid-based techniques for detection of infectious agents, such as influenza viruses, is that of nucleic acid sequence variation. DNA probes, 70-nt long, some including the nucleotide analog deoxyribose-Inosine (dInosine), were analyzed for hybridization tolerance to different amounts and distributions of mismatching bases, e.g. synonymous mutations, in target DNA. Microsphere-linked 70-mer probes were hybridized in 3M TMAC buffer to biotinylated single-stranded (ss) DNA for subsequent analysis in a Luminex® system. When mismatches interrupted contiguous matching stretches of 6 nt or longer, it had a strong impact on hybridization. Contiguous matching stretches are more important than the same number of matching nucleotides separated by mismatches into several regions. dInosine, but not 5-nitroindole, substitutions at mismatching positions stabilized hybridization remarkably well, comparable to N (4-fold) wobbles in the same positions. In contrast to shorter probes, 70-nt probes with judiciously placed dInosine substitutions and/or wobble positions were remarkably mismatch tolerant, with preserved specificity. An algorithm, NucZip, was constructed to model the nucleation and zipping phases of hybridization, integrating both local and distant binding contributions. It predicted hybridization more exactly than previous algorithms, and has the potential to guide the design of variation-tolerant yet specific probes.
authors.affiliation: Clinical Virology, Department of Medical Sciences, Uppsala University and Academic Hospital, 751 85 Uppsala, Sweden.
authors.forename: Christina
authors.initials: C
authors.lastname: Ohrmalm
authors.forename: Magnus
authors.initials: M
authors.lastname: Jobs
authors.forename: Ronnie
authors.initials: R
authors.lastname: Eriksson
authors.forename: Sultan
authors.initials: S
authors.lastname: Golbob
authors.forename: Amal
authors.initials: A
authors.lastname: Elfaitouri
authors.forename: Farid
authors.initials: F
authors.lastname: Benachenhou
authors.forename: Maria
authors.initials: M
authors.lastname: Strømme
authors.forename: Jonas
authors.initials: J
authors.lastname: Blomberg
mesh_terms: D000465:Algorithms; D020137:Base Pair Mismatch; D001483:Base Sequence; D002648:Child; D015342:DNA Probes / Q000737:chemistry*; D006801:Humans; D007211:Indoles / Q000737:chemistry; D007288:Inosine / Q000031:analogs & derivatives* / Q000737:chemistry; D009693:Nucleic Acid Hybridization / Q000379:methods*; D012401:Rotavirus / Q000235:genetics / Q000302:isolation & purification; D013696:Temperature
chemical_list: D015342:DNA Probes; D007211:Indoles; D007288:Inosine; C012271:deoxyinosine; C090067:5-nitroindole