Rapid, single-step nucleic acid detection.
A rapid detection method for nucleic acid based on bioluminescence resonance energy transfer (BRET) from the luminescence donor Renilla luciferase to an acceptor quantum dot upon oligonucleotide probe hybridization has been developed. Utilizing a competitive assay, we detected the target nucleic acid by correlating the BRET signal with the amount of target present in the sample. This method allows for the detection of as little as 4 pmol (20 nM) of nucleic acid in a single-step, homogeneous format both in vitro in a buffer matrix as well as in a cellular matrix. Using this method, one may perform nucleic acid detection in as little as 30 min, showing much improvement over time-consuming blotting methods and solid-phase methods which require multiple wash steps to remove unbound probe. This is the first report on the use of quantum dots as a BRET acceptor in the development of a nucleic acid hybridization assay.
Cissell
Kyle A
KA
Department of Chemistry and Chemical Biology, Indiana University Purdue University Indianapolis, 402 N. Blackford St., Room LD 326, Indianapolis, IN 46202, USA.
Campbell
Sean
S
Deo
Sapna K
SK
D004247:DNA / Q000032:analysis*; D004735:Energy Transfer; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D007093:Imidazoles / Q000737:chemistry; D049410:Luciferases, Renilla / Q000737:chemistry*; D049408:Luminescent Agents / Q000737:chemistry*; D008163:Luminescent Measurements / Q000379:methods*; D009693:Nucleic Acid Hybridization / Q000379:methods; D015345:Oligonucleotide Probes / Q000737:chemistry; D011719:Pyrazines / Q000737:chemistry; D045663:Quantum Dots
D007093:Imidazoles; D049408:Luminescent Agents; D015345:Oligonucleotide Probes; D011719:Pyrazines; C017144:coelenterazine; D004247:DNA; D049410:Luciferases, Renilla