Mutation detection and single-molecule counting using isothermal rolling-circle amplification

In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes

“…14,16,27,28 We found that the use of rubber silicone reaction chambers greatly improved the efficiency of the RCA reaction. Therefore, we performed the RCA reactions on dehydrated slides in 50 mM Tris-HCl pH 7.5, 10 mM MgCl 2 , 20 mM (NH 4 ) 2 SO 4 , 0.2 mg/ml BSA, 1 mM DTT, 0.25 mM dNTP, 10% glycerol and 1 U/ml F29 DNA polymerase (Fermentas) at 37 1C for 30 min in the silicone mask reaction chambers placed on a shaking platform, except for the tonsillar tissue section, where the RCA was carried out under a coverslip.…”

“…If, instead, the reaction is set up so that the RCA template can form only in a target-specific circularization reaction in situ, nonspecific signals should be greatly diminished; and promising results have been reported with this approach 14,15 . Regrettably, replication of circular probes that remain wound around their target strands seems to be inhibited, probably as a consequence of molecular crowding 16 , although there is some controversy in the literature about this topological inhibition 17 .…”

Isothermal Strand-Displacement Amplification Applications for High-Throughput Genomics

“…the generation of tandem repeat libraries and as reporter constructs 1,4 . Rolling-circle amplification has recently been employed using exonuclease resistant random hexamer primers and Φ29 DNA polymerase to generate large quantities of DNA from traditional vectors in an isothermal reaction by way of multiply-primed template and DNA strand displacement 2 .…”

Garbage in, garbage out: A critical evaluation of strategies used for validation of immunohistochemical biomarkers

“…The PLA technique is highly sensitive method determining protein interactions and analysing post‐translational modifications (Blokzijl et al., 2010; Lizardi et al., 1998; Soderberg et al., 2006). It is based on the principle that two or more oligonucleotide‐conjugated antibodies need to bind in close proximity in order to detect a signal, and can be utilized directly in frozen or FFPE tissue sections (Soderberg et al., 2008; Zieba et al., 2010).…”