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.