The 5′ and 3′ arm sequences of the molecular beacons were designed to form a stable hybrid at 5 to 10°C above the annealing temperature of

the PCR assay. After selecting three slightly different probe and arm sequences, the molecular Epigenetics inhibitor beacon for recA amplicon were optimized. These probes were labeled with a Fluorescein (FAM) reporter molecule at their 5′ terminals and Black Hole Quencher 1 (BHQ-1) or dabcyl at their 3′ terminals. Using similar parameters, a nidogen specific molecular beacon was also designed. KU55933 The Nidogen molecular beacon was labeled with a 5′ Tetrachlorofluorescein (TET) reporter molecule and a 3′ BHQ-1 quencher. The fluorophores and quenchers were chosen based on the specifications of the spectrofluorometric thermal cycler platform on which the assays were carried out. The sequences of the molecular beacons used in this study are listed in Table 1. RG7112 cost A detailed protocol for the synthesis and purification of molecular beacons can be found at http://​www.​molecular-beacons.​org. PCR products were detected by fluorescence measurement by including SYBR Green I dye (Molecular Probes, OR) or molecular beacons in the assays. The amplification program for SYBR Green I consisted of heating at 95°C for 2 minutes, followed

by 50 cycles of heating at 95°C for 15 s, annealing at 60°C for 30 s, polymerization at 72°C for 20 s and fluorescence detection at 80°C for 10 s. Mouse nidogen was amplified similarly except that fluorescence was detected at 82°C instead of 80°C. For PCR assays using molecular beacon probes, 200 nM of RecA or Nidogen molecular beacon were included per reaction. The amplification program consisted of heating at 95°C for 2 minutes, followed by 50 cycles of heating at 95°C for 15 s, annealing and fluorescence detection at 60°C for 30 s, and polymerization at 72°C for 20 s. Determination Prostatic acid phosphatase of thermal denaturation profiles

In order to determine the melting temperatures of the molecular beacon stem and the molecular beacon probe-target hybrid, a denaturation profile analysis was carried out. For each probe, three tubes containing 200 nM molecular beacon, 3 mM MgCl2, 50 mM KCl, and 10 mM Tris-HCl (pH 8.0), in a 50-μl volume were prepared. A two-fold molar excess of an oligonucleotide that is complementary to the molecular beacon probe sequence, a two-fold excess of an oligonucleotide unrelated to the probe sequence, or only buffer were added in these tubes. The fluorescence of each solution was determined as a function of temperature. The thermal cycler was programmed to decrease the temperature of the solutions from 80°C to 30°C in 1°C steps, with each step lasting 1 min, while monitoring fluorescence during each step. Acknowledgements We thank John M. Leong, Sanjay Tyagi and Diana Palmeri for careful review of the manuscript.