V. Shick, S. Dubiley, N. Kalganova, D. Pobedimskaya, D. Prudnikov, A. Perov, D. Guschin, S. Belikov, and A. Mirzabekov
Argonne National Laboratory (U.S.A.) and Engelhardt Institute of Molecular Biology (Moscow)-Joint Human Genome Program.
Oligonucleotide microchips have been tested as diagnostics of genetic hybridization diseases. Several methods have been evaluated for the preparation of hybridization probes. Double-stranded DNA shows much lower efficiency of hybridization with the microchips than does single-stranded DNA. The efficiency is increased significantly by fragmentation of the dsDNA into short pieces. Different methods of preparation of ssDNA have been tested, such as asymmetric PCR, isolation of PCR biotin-ssDNA on Dynabeads, and immobilization of denatured PCR dsDNA on a resin followed by DNA synthesis with a DNA primer. Long ssDNA fragments may form hairpin structures and diffuse slowly into the gel to interact with the gel-immobilized oligonucleotides; they are poorly hybridized with the microchips. DNA fragmentation significantly increased the hybridization efficiency. Enzymatic and chemical random fragmentation of DNA have been compared. Chemical and enzymatic methods of introducing fluorescent labels into DNA fragments for monitoring of the hybridization on the microchips by fluorescent microscopy have been developed. The use of PCR with T7 RNA promoter containing primers and the following transcription with T7 RNA polymerase have also been tested for preparing RNA for microchip hybridization. The effect of different conditions of hybridization in a low-volume hybridization chamber have been tested. A reliable identification of heterozygous and homozygous beta-thalassemia mutations has been demonstrated by hybridization with the microchips of the probes prepared by these methods.
*Work supported in part by the U.S. Department of Energy, Office of Health and Environmental Research, under Contract No. W-31-109-ENG-38 and Russian Human Genome Program.
The submitted manuscript has been authorized by a contractor of the U.S. Government under contract No. W-31-109-ENG-38. Accordingly, the U.S. Government retains a nonexclusive, royalty-free license to publish or reproduce the published form of this contribution, or allow others to do so, for U.S. Government purposes.