Hybridization properties of long nucleic acid probes for detection of variable target sequences, and development of a hybridization prediction algorithm.
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.
Ohrmalm
Christina
C
Clinical Virology, Department of Medical Sciences, Uppsala University and Academic Hospital, 751 85 Uppsala, Sweden.
Jobs
Magnus
M
Eriksson
Ronnie
R
Golbob
Sultan
S
Elfaitouri
Amal
A
Benachenhou
Farid
F
Strømme
Maria
M
Blomberg
Jonas
J
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
D015342:DNA Probes; D007211:Indoles; D007288:Inosine; C012271:deoxyinosine; C090067:5-nitroindole