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The electronic form of this document may be cited in the following style:
Human Genome Program, U.S. Department of Energy, DOE Human Genome Program Contractor-Grantee Workshop IV, 1994.
Abstracts scanned from text submitted for November 1994 DOE Human Genome Program Contractor-Grantee Workshop. Inaccuracies have not been corrected.
Chemiluminescent Detection of Multiplex Labeled Microsatellite Markers and DNA Sequences
Chris S. Martin, Laurie Butler, Greg Schneider and Irena Bronstein
Tropix, Inc., Bedford, MA 01730
We have developed a technique for sequential nonisotopic detection of multiple sets of DNA reaction products which are labeled with different haptens. This multiplex labeling approach utilizes hapten-specific alkaline phosphatase conjugates and chemiluminescent 1,2-dioxetane substrates. The chemiluminescent detection method generates intense light signals which can be easily imaged with standard X-ray film. For DNA sequencing, multiple primers, each with a unique ligand label, are incorporated in sequencing reactions, the products are separated, transferred to nylon membrane and detected by binding hapten specific alkaline phosphatase conjugates. We have used primers labeled with biotin, digoxigenin, fluorescein and 2,4-dinitrophenyl (DNP), enabling the acquisition of four images of DNA sequence data from a single nylon membrane. The need for large scale screening of polymorphic microsatellite markers for genetic mapping led us to adapt this technique for the detection of PCR amplified microsatellite markers. Individual sets of PCR primers labeled with each hapten are utilized to amplify different microsatellite repeat markers. The amplified markers for each genomic DNA sample are loaded in a single gel lane, electrophoretically separated, transferred to a nylon membrane and detected sequentially with hapten-specific alkaline phosphatase conjugates. Each of the four different haptens have been used for three amplimer pairs, to generate three different size fragments with each label. Thus, 12 different markers can be typed from a single gel lane. In addition, we have evaluated a new 1,2-dioxetane substrate, CDP-Star(tm), which generates extremely intense light signals. This high light output enables efficient detection of the chemiluminescent signals with low light sensitive CCD cameras and rapid acquisition of digitized images of the data suitable for automated analysis is possible. Chemiluminescent detection of overlapping sets of DNA fragments coupled with multiplex labeling is an efficient, non-radioactive method for PCR product detection and DNA sequence analysis.
This work was funded by the DOE Genome Program. Contract No. DE FG05 92ER81389