A homogeneous DNA hybridization system by using a new luminescence terbium chelate.
Homogeneous DNA hybridization assay based on the luminescence resonance energy transfer (LRET) from a new luminescence terbium chelate, N,N,N(1),N(1)-[2,6-bis(3'-aminomethyl-1'-pyrazolyl)-4-phenylpyridine]tetrakis(acetic acid) (BPTA)-Tb(3+) (lambda(ex) = 325 nm and lambda(em) = 545 nm) to an organic dye, Cy3 (lambda(ex) = 548 nm and lambda(em) = 565 nm), has been developed. In the system, two DNA probes whose sequences are complementary to the two different consecutive sequences of a target DNA are used; one of the probes is labeled with the Tb(3+) chelate at the 3'-end, and the other is with Cy3 at the 5'-end. Labeling of the Tb(3+) chelate is accomplished via the linkage of a biotin-labeled DNA probe with the Tb(3+) chelate-labeled streptavidin. Strong sensitized emission of Cy3 was observed upon excitation of the Tb(3+) chelate at 325 nm, when the two probe DNAs were hybridized with the target DNA. The sensitivity of the assay was very high compared with those of the previous homogeneous-format assays using the conventional organic dyes; the detection limit of the present assay is about 30 pM of the target DNA strand.
Sueda
Shinji
S
Department of Chemistry, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan.
Yuan
Jingli
J
Matsumoto
Kazuko
K
D000085:Acetates / Q000737:chemistry*; D001483:Base Sequence; D015342:DNA Probes / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004735:Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D007700:Kinetics; D008163:Luminescent Measurements; D015394:Molecular Structure; D009693:Nucleic Acid Hybridization / Q000379:methods*; D011720:Pyrazoles / Q000737:chemistry*; D011725:Pyridines / Q000737:chemistry*; D012680:Sensitivity and Specificity; D013194:Staining and Labeling; D019809:Streptavidin / Q000737:chemistry; D013725:Terbium / Q000737:chemistry*
D000085:Acetates; D015342:DNA Probes; D005456:Fluorescent Dyes; C428807:N, N,N1,N1-(2,6-bis(3'-aminomethyl-1'-pyrazolyl)-4-phenylpyridine)tetrakis(acetic acid); D011720:Pyrazoles; D011725:Pyridines; D013725:Terbium; D019809:Streptavidin