2.1 Synthesis and purification of oligonucleotides
The oligonucleotides B-MBPup, (5’-Biotin CGGCGATAAAGGCTATAACGG), MBPup (CGGCGATAAAGGCTATAACGG), B-MBPR1 (5’-Biotin CTGGAACGCTTTGTCCGGGG), MBPR1 (5’- CTGGAACGCTTTGTCCGGGG) and oligo-loop, (5’- TTTTTTTTTTTTTTTTTTTGCTGGAATTCGTCAGACTGGCCGTCGTTTTACAACGGAACG TTGT AAAACGACGGC), were all synthesized and HPLC purified by MWG Biotech (High Points, NC, USA).
2.2 Template preparation
The biotinylated PCR products were immobilized onto streptavidin-coated superparamagnetic beads, Dynabeads™ M280-Streptavidin (Dynal A.S., Oslo, Norway). Single-stranded DNA (ssDNA) was obtained by removing the supernatant after incubation of the immobilized PCR product in 100 mM NaOH for 3 min. Sequencing primers MBP-up or MBP-R1 (5 pmol) were hybridized to the immobilized ssDNA strand that was obtained from an amplified product via polymerase chain reaction (PCR).
2.3 BRC assay
The immobilized single stranded PCR product was resuspended in annealing buffer (10mM Tris-acetate pH 7.75, 2mM Mg-acetate), and primers were added to the single stranded templates. Hybridization of the template and primers was performed (by incubation at 95°C for 3 min, 72°C for 5min and then cooling to room temperature) and extension conducted after the addition of exonuclease-deficient (exo-) Klenow DNA polymerase (10 U; Fermentas, NY, USA) and all 4 nucleotide triphosphates (1.4 mM nucleotides (1.4mM final concentration) to the extension mixture. After completion of the reaction, the content of each well was serially diluted for comparison of extension analysis (PPi concentration). Extension and real-time luminometric monitoring was performed at either 37°C in an IVIS™ imaging system (Xenogen, Alameda, CA USA), or at 27°C in an Lmax™ microplate luminometer (Molecular Devices, Sunnyvale, CA, USA). A luminometric reaction mixture was added to the substrate at different concentrations (extended primed single-stranded DNA or self primed oligonucleotide). The 40µl bioluminescence assay mixture contained: 3.0 µg luciferase (Promega, USA), 50 mU recombinant ATP-sulfurylase (Sigma, USA), 0.1 M Tris-acetate (pH 7.75), 0.5 mM EDTA (Sigma, USA), 5 mM Mg-acetate (Sigma, USA), 0.1% (w/v) bovine serum albumin (Sigma, USA), 2.5 mM dithiothreitol (Sigma, USA), 10 µM adenosine 5’-phosphosulfate (APS) (Biolog, Belgium), 0.4 mg polyvinylpyrrolidone/ml (molecular weight 360000) and 100 µg D-luciferin/ml (BioThema, Uppsal, Sweden), and the emitted light was detected in real time and measured after approximately 45 sec with 1 sec and 10 sec integration times for the CCD imaging system and luminometer, respectively.
2.4 Detection Devices
To estimate the quantity of the target nucleic acid, we counted the photons generated by the BRC process. The general luciferase generation of photons has a quantum efficiency (Q.E.) of approximately 0.88 per consumed ATP molecule, and the maximum wavelength (which depends on the type of luciferase), is in the visible range of the optical spectrum (e.g., 565 nm for firefly luciferase).
A variety of photosensitive devices have been developed to detect bioluminescent signals, and these have been used to detect light from the regenerative phenomenon. These devices include photomultiplier tubes (PMTs), charge coupled devices (CCDs), and photodiodes. The photosensitive device can either be in close proximity to the BRC reaction to receive the incident photons directly, or at a distance from the reaction buffer with a light coupling device (e.g. optical fiber or mirror system) to convey photons from the sample to the detector.
In our experiments we used both a cooled CCD camera imaging system (IVIS; Xenogen) and a luminometer (Lmax™; Molecular Devices) that employed a single PMT detector. The light coupling efficiencies of each system (including path loss), from the microtiter plate where the DNA samples were located, to the sensor, were approximately 0.012% and 8% for the CCD and PMT systems, respectively, as shown in .
(a) CCD camera system which simultaneously measures the light from reference buffer and target sample, and (b) PMT imaging system in luminometer.
In the CCD imaging system a 96-well microtiter plate with multiple DNA samples was placed 18 cm below the lens of the camera (), and in the luminometer a 384-well microtiter plate was inserted in the instrument chamber, where a PMT directly moves into close proximity (1cm) of the sample for reading ().