Bioluminescence Assay

Protocol 1

The 45ul Bioluminescence assay mixture contained: 0.1M
Tris-acetate (pH7.75), 0.5mM EDTA (Sigma,USA), 5 mM
Mg-acetate (Sigma, USA), 0.1%(w/v) bovine serum albu-
min (Sigma, USA), 2.5 mM dithiothreitol (Sigma, USA),
2ul of exonuclease-deficient Klenow DNA polymerase (10
U/ul; Fermentas, NY, USA) 10uM adnosine 5’-phosphosulfate
(APS) (Biolog, Germany), 0.4mg/ml polyvinylpyrrolidone
(MW 36000), 100ug/ml D-luciferin (BioThema, Uppsala,
Sweden), 3.0ug luciferase (Promega, USA) and 50mU re-
combinant ATP-sulfurylase (Sigma, USA) or recombinant
thermo-stable ATP-sulfurylase (from Aquifex aeolicus, pu-
rified in Panoroma Research Inc., USA). 10pmol of oligo-
loop diluted in 0.1M Tris-acetate (pH7.75) was added to the
reaction mix, and subsequently to generate the biolumines-
cence light, 2ul of all 4 nucleotides (dCTP, dGTP, dTTP and
dATP « S) with 1.4 mM final concentration was injected into
the reaction mix. The emitted photons were measured using
H5783-03 PMT (Hamamatsu, Japan).

Protocol 2

An aliquot of synthesized cDNA was added to 50 μl of reaction mixture (see Ronaghi et al., Anal. Biochem. 242:84–89, 1996 with modifications) containing 250 ng luciferase (Promega, Madison, Wis.), 50 mU ATP sulfurylase (Sigma Chemical Co., St. Louis, Mo.), 2 mM dithiothreitol, 100 mM Tris-Acetate pH 7.75, 0.5 mM EDTA, 0.5 mg BSA, 0.2 mg polyvinylpyrrolidone (M_r 360.000), 10 μg D-luciferin (Biothema, Dalaro, Sweden), 5 mM magnesium acetate and 10 attomole to 0.01 attomole purified pyrophosphate or ATP. The addition of very low amounts of pyrophosphate or ATP (or analogs) was found to be important to decrease background light emission from the reaction mixture. Although the precise mechanism is unknown, BRC performed without adding small amounts of ATP or PPi consistently exhibited background luminescence that precluded accurate measurement of target nucleic acids present in amounts of about a femtomole or lower. Inorganic pyrophosphate present in the cDNA sample as a result of polymerase mediated dNTP incorporation was converted to ATP by sulfurylase. The ATP was used to generate light in a luciferin/luciferase reaction.