Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1993-10-22
1995-11-28
Fleisher, Mindy B.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 71, 435 75, 435 79, C12Q 168
Patent
active
054707102
ABSTRACT:
A method is disclosed for automated multiplex sequencing of DNA with an integrated automated imaging hybridization chamber system. This system comprises an hybridization chamber device for mounting a membrane containing size-fractionated multiplex sequencing reaction products, apparatus for fluid delivery to the chamber device, imaging apparatus for light delivery to the membrane and image recording of fluorescence emanating from the membrane while in the chamber device, and programmable controller apparatus for controlling operation of the system. The multiplex reaction products are hybridized with a probe, then an enzyme (such as alkaline phosphatase) is bound to a binding moiety on the probe, and a fluorogenic substrate (such as a benzothiazole derivative) is introduced into the chamber device by the fluid delivery apparatus. The enzyme converts the fluorogenic substrate into a fluorescent product which, when illuminated in the chamber device with a beam of light from the imaging apparatus, excites fluorescence of the fluorescent product to produce a pattern of hybridization. The pattern of hybridization is imaged by a CCD camera component of the imaging apparatus to obtain a series of digital signals. These signals are converted by the controller apparatus into a string of nucleotides corresponding to the nucleotide sequence an automated sequence reader. The method and apparatus are also applicable to other membrane-based applications such as colony and plaque hybridization and Southern, Northern, and Western blots.
REFERENCES:
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Richterich et al. (1989) Bio Techniques, vol. 7, pp. 52-59.
Cano et al. (1992) Bio Techniques, vol. 12, pp. 264-268.
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Holtke et al. (1990) Nucleic Acids Research, vol. 18, pp. 5843-5851.
Cherry Joshua L.
Di Sera Leonard J.
Dunn Diane M.
Ferguson F. Mark
Gesteland Raymond F.
Carter Philip W.
Fleisher Mindy B.
University of Utah
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