Chris S. Martin, John C. Voyta 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. 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 antibody 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. While satisfactory chemiluminescent images can be obtained on nylon membranes, there is a need for improvement of the membrane support used for chemiluminescent detections. Recently, we developed membranes which incorporate a polymer enhancer surface layer. These membranes, prepared by overcoating certain membrane supports, exhibit greater chemiluminescence signal intensities and lower background noise. These membranes were tested by performing chemiluminescent detection of multiple labeled oligonucleotides or DNA sequence ladders. The development of a superior membrane would enable more rapid imaging of DNA sequences on x-ray film or electronic imaging devices (i.e. CCD cameras).
This work was funded by the DOE Genome Program. Contract No. DE FG05 92ER81389
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