Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1998-11-04
1999-07-06
LeGuyader, John L.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 912, 536 231, 536 243, 536 253, 536 2532, C12Q 168, C07H 2104, C12P 1934
Patent
active
059196300
ABSTRACT:
Single-stranded signal primers are modified by linkage to two dyes which form a donor/acceptor dye pair. The two dyes are positioned in sufficiently close spatial proximity on the signal primer that the fluorescence of the first dye is quenched by the second dye. The signal primer may further comprise a restriction endonuclease recognition site (RERS) between the two dyes. As the signal primer is initially single-stranded and remains single-stranded in the absence of target, the restriction endonuclease recognition site is not cleavable or nickable by the restriction endonuclease. In the presence of target, however, signal primer and the restriction endonuclease recognition site are rendered double-stranded and cleavable or nickable by the restriction endonuclease. Cleavage or nicking separates the two dyes and a change in fluorescence due to decreased quenching is detected as an indication of the presence of the target sequence or of target sequence amplification.
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L.G. Lee, et al., "Allelic Discrimination by Nick-translation PCR with Fluorogenic Probes" Nucl. Acids Res. 21:3761-3766 (1993).
S.P. Lee, et al. "A Fluorometric Assay for DNA Cleavage Reactions Characterized with BamHl Restriction Endonuclease" Anal. Biochem. 220:377-383 (1994).
S.S. Ghosh, et al. "Real Time Kinetics of Restriction Endonuclease Cleavage Monitored by Fluorescnece Resonance Energy Transfer" Nucl. Acids Res. 22:3155-3159 (1994).
Linn C. Preston
Nadeau James G.
Pitner J. Bruce
Schram James L.
Vonk Glenn P.
Becton Dickinson and Company
Fugit Donna R.
LeGuyader John L.
Shibuya Mark L.
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