The probes were 106–123 nucleotides (nt) in length, consisting of two adjacent target complementary sequences with a 48 nt linker region (Figure 1). To optimise binding to target DNA, probes were designed with a minimum of secondary structure and with a Tm of the 5′-end probe binding arm greater than the temperature used for probe ligation (62°C; see below). To increase the specificity, the 3′-end binding arm was designed to have a Tm (51–56°C) below the ligation temperature
[25]. In particular, careful attention was paid to the linker region for each point mutation-specific probe to (i) minimise similarity to those mutations closely-located to the mutation HDAC inhibitor of interest and (ii) to allow primer binding during RCA and amplification of the probe-specific signal. The 2 primers used for RCA – RCA primer 1 (5′ ATGGGCACCGAAGAAGCA 3′, Tm 55°C) and RCA primer 2 (5′ CGCGCAGACACGATA 3′, Tm 55°C) – were designed to specifically bind the linker region of the probes (Additional file 1) Purification of RCA template Prior to ligation Smoothened Agonist of the probe, ERG11 PCR products were purified to remove excess buffer, dNTP and primers: 25 μl of
the PCR product was added to a well of a Millipore PCR purification plate (Pall Life Sciences, Ann Arbor, MI, USA) which was then placed on a vacuum manifold for 10–20 min to draw fluid and small particles through the membrane, leaving DNA on top of the membrane. A further 25 μl of dH2O was added to the well and the process repeated. The plate was removed from the vacuum, 20 μl of dH2O was added and the mixture incubated at 25°C for 2 min before transferring to a clean Eppendorf tube. Purified PCR products were stored at 4°C. Ligation of padlock probe and exonucleolysis Purified amplified PCR product (1011 copy numbers of DNA template [DNA calculator; http://www.uri.edu/research/gsc/resources/cndna.html])
else was mixed with 2 U of Pfu DNA ligase (Stratagene, La Jolla, CA, USA) and 0.1 μM padlock probe as previously described [25] and subjected to multiple cycle ligation comprising one cycle of denaturation at 94°C for 5 min, followed by five cycles at 94°C for 30 s and 4 min of ligation at 62°C. Exonucleolysis was then performed to remove Evofosfamide solubility dmso unligated probe and template PCR product; the purpose of the last step is to reduce subsequent ligation-independent amplification events during RCA. It was performed in 20-μl volumes by adding 10 U each of exonuclease I and exonuclease III (New England Biolabs, UK) to the ligation mixture and incubating at 37°C for 60 min followed by 95°C for 3 min.