Imaging of DNA sequences with chemiluminescence.

Abstract

We have coupled a chemiluminescent detection method that uses an alkaline phosphatase label to the genomic DNA sequencing protocol of Church and Gilbert

Real-time detection of DNA hybridization and melting on oligonucleotide arrays by using optical wave guides.

“…Most of the DNA hybridization detection methods employed thus far use radioactive (15)(16)(17)(18)(19)(20), enzyme-based chemiluminescent (21), or fluorescent (22) labels. Detection and measurement can be accomplished with phosphor systems (23) or, for the latter two labels, with charge-coupled device (CCD) cameras (24), cooled CCD cameras, image intensifiers coupled to CCD cameras, or a photomultiplier tube coupled with mechanical raster scanning (12,25).…”

Chemiluminescence detection of proteins from single cells.

“…The high protein-binding capacity of these membranes, which substantially increased the background, mandated an unusually effective blocking agent. As in other chemiluminescence detection methods (13,14), casein proved to be the most effective single blocking agent. We initially used as a blocking solution 4% casein and 2% hemoglobin, a combination that improved the signal/ noise ratio substantially over 6% casein or 6%o hemoglobin alone.…”

“…1 also shows that treatment of the blocking and streptavidin-dilution solutions with avidin-agarose improved detection by a factor of -8; 500 fg of biotinylated BSA was the least that could be detected if the casein blocking solution had not been treated. Although the output of light remained substantial for at least 20 hr (14), the membranes of Fig. 1 immune detection on slot blots of the microtubule motor protein kinesin (27,28).…”

“…Nylon membranes are advantageous in that they enhance light production in the assay procedure (14). Uncharged nylon membranes are of limited use for total protein detection, for proteins of widely disparate molecular size cannot be quantitatively transferred to and retained by such membranes (23).…”

“…Chemiluminescence methods based on horseradish peroxidase (11) and alkaline phosphatase (12)(13)(14) have been developed for the detection of very small amounts of DNA. Adaptation of these assays to protein detection has been impeded, however, by the high background that characteristically accompanies the signal.…”