Effect of unlabeled helper probes on detection of an RNA target by bead-based sandwich hybridization
2004 (English)In: BioTechniques, ISSN 0736-6205, Vol. 36, no 1, 124-+ p.Article in journal (Refereed) Published
Unlabeled helper oligonucleotides assisting a bead-based sandwich hybridization assay were tested for the optimal placement of the capture and detection probes. The target used was a full-length in vitro synthesized mRNA molecule. Helper probes complementary to regions adjacent to the binding site of the 5' end attached capture probe were found much more effective than helper probes targeting positions adjacent to the detection probe binding site. The difference is believed to be caused by a disruption of the RNA secondary structure in the area where the capture probe binds, thereby reducing structural interference from the bead. The use of additional helpers showed an additive effect. Using helpers, at both sides of the capture and detection probes showed a 15- to 40-fold increase in hybridization efficiency depending on the target, thereby increasing the sensitivity of the hybridization assays. Using an electrical chip linked to the detection probe for the detection of p-ominophenol, which is produced by alkaline phosphatase, a detection limit of 2 x 10(-13) M mRNA molecules was reached without the use of a nucleic acid amplification step.
Place, publisher, year, edition, pages
2004. Vol. 36, no 1, 124-+ p.
single-stranded-dna, 16s ribosomal-rna, labeled oligonucleotide probes, in-situ accessibility, modular primers, stacking, hexamers, capture, arrays
Biochemistry and Molecular Biology
IdentifiersURN: urn:nbn:se:kth:diva-23091ISI: 000188083600017ScopusID: 2-s2.0-0346024108OAI: oai:DiVA.org:kth-23091DiVA: diva2:341789
QC 20100525 QC 201111012010-08-102010-08-102011-11-01Bibliographically approved