Signal amplification of padlock probes by rolling circle replication

• Published in: Nucleic acids research Vol. 26; no. 22; pp. 5073 - 5078
• Main Authors: Banér, Johan, Nilsson, Mats, Mendel-Hartvig, Maritha, Landegren, Ulf
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 15.11.1998
• Subjects: Base Sequence; DNA Replication; DNA, Circular - chemistry; DNA, Circular - genetics; Models, Molecular; Molecular Probe Techniques; Nucleic Acid Conformation; Oligonucleotide Probes - chemical synthesis; Oligonucleotide Probes - chemistry; Oligonucleotide Probes - genetics
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/26.22.5073

Circularizing oligonucleotide probes (padlock probes) have the potential to detect sets of gene sequences with high specificity and excellent selectivity for sequence variants, but sensitivity of detection has been limiting. By using a rolling circle replication (RCR) mechanism, circularized but not unreacted probes can yield a powerful signal amplification. We demonstrate here that in order for the reaction to proceed efficiently, the probes must be released from the topological link that forms with target molecules upon hybridization and ligation. If the target strand has a nearby free 3′ end, then the probe-target hybrids can be displaced by the polymerase used for replication. The displaced probe can then slip off the target strand and a rolling circle amplification is initiated. Alternatively, the target sequence itself can prime an RCR after its non-base paired 3′ end has been removed by exonucleolytic activity. We found the Φ29 DNA polymerase to be superior to the Klenow fragment in displacing the target DNA strand, and it maintained the polymerization reaction for at least 12 h, yielding an extension product that represents several thousand-fold the length of the padlock probe.