Automated hybridization/imaging device for fluorescent multiplex DNA sequencing
First Claim
1. A method for enzyme-linked fluorescence detection of nucleic acid comprising the steps of:
- (a) attaching the nucleic acid to be detected to a nylon membrane;
(b) contacting the nylon membrane with an oligonucleotide probe containing a binding moiety for binding alkaline phosphatase and which is able to hybridize specifically with a complementary sequence on the nucleic acid, thereby hybridizing said probe to said nucleic acid;
(c) contacting the probe with alkaline phosphatase, thereby binding the alkaline phosphatase to said binding moiety on the probe;
(d) contacting the alkaline phosphatase with BBTP so that the alkaline phosphatase converts the BBTP into a fluorescent product, wherein the fluorescent product interacts with the nylon membrane and is spatially localized thereon; and
(e) illuminating said fluorescent product with a beam of light capable of exciting fluorescence by said fluorescent product, thereby exciting fluorescence by said fluorescent product wherein said fluorescence produces a pattern that reflects the location of said nucleic acid on said membrane.
4 Assignments
0 Petitions
Accused Products
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.
-
Citations
10 Claims
-
1. A method for enzyme-linked fluorescence detection of nucleic acid comprising the steps of:
-
(a) attaching the nucleic acid to be detected to a nylon membrane; (b) contacting the nylon membrane with an oligonucleotide probe containing a binding moiety for binding alkaline phosphatase and which is able to hybridize specifically with a complementary sequence on the nucleic acid, thereby hybridizing said probe to said nucleic acid; (c) contacting the probe with alkaline phosphatase, thereby binding the alkaline phosphatase to said binding moiety on the probe; (d) contacting the alkaline phosphatase with BBTP so that the alkaline phosphatase converts the BBTP into a fluorescent product, wherein the fluorescent product interacts with the nylon membrane and is spatially localized thereon; and (e) illuminating said fluorescent product with a beam of light capable of exciting fluorescence by said fluorescent product, thereby exciting fluorescence by said fluorescent product wherein said fluorescence produces a pattern that reflects the location of said nucleic acid on said membrane. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
-
Specification