Chemical Ways to Improve DNA Sequencing by Hybridization with Oligonucleotide Microchips (SHOM)*

Edward N. Timofeev, Andrew G. Kunitsyn, Svetlana V. Kochetkova, Andrew D. Mirzabekov, and Vladimir L. Florentiev

Argonne National Laboratory (U.S.A.) and Engelhardt Institute of Molecular Biology (Moscow)-Joint Human Genome Program.

Sequencing DNA by using the hybridization with oligonucleotide microchips (SHOM) method is one of the prospective methods for DNA sequencing and mutation detection.[1,2] To improve this method, we have carried out the following investigations: (i) development of more effective procedures for oligonucleotide immobilization (ii) development of an approach to increase the selectivity of hybridization and (iii) study of the physico-chemical basis of DNA hybridization on the microchip. To increase the stability of oligonucleotide binding to the gel, we have elaborated a number of alternative methods for activation of the gel. These techniques are based on copolymerization of acrylamide with acrylic acid derivatives containing either hydrazide or amino groups. Immobilization of oligonucleotides on copolymers was carried out by the three following methods:

• (A) interaction of hydrazide copolymer with an dialdehyde derivative of the oligonucleotide;
• (B) treatment of amino copolymer with an dialdehyde derivative of the oligonucleotide in the presence of reductant (pyridine-borane complex), which results in the formation of a very stable amino bond;
• (C) reaction of amino copolymer with an oligonucleotide derivative containing an activated ester (N-hydroxysuccinimide ester) coupled through a linker to oligomers. In the latter case, the binding of oligomers is stronger than in Method A but not as strong as in Method B.

To increase the selectivity of hybridization, we have studied the hybridization properties of oligonucleotides in which the universal base analog 5-nitroindole was added to the end of the sense sequence. Hybridization experiments showed that discrimination between perfect duplexes and duplexes with terminal mismatches was increased.

Earlier we proposed a theoretical model for the dissociation of the duplex formed by DNA with immobilized oligonucleotides.[3] To apply this model practically for the calculation of relatively stability of duplexes, we have performed an experimental study of the thermodynamic parameters of such duplex formation. The results obtained enable us to prepare the "normalized" matrix (the matrix with equalized stability of duplexes having GC-contents).

*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.

[1] Khrapko, K.R., Lysov, Yu.P., Khorlin, A.A., Shick, V.V., Florentiev, V.L., Mirzabekov, A.D. FEBS Lett., 256, 118-122 (1989).

[2] Khrapko, K.R., Lysov, Yu.P., Khorlin, A.A., Ivanov, I.B., Yershov, G.M., Vasilenko, S.K., Florentiev, V.L., and Mirzabekov, A.D. J. DNA Sequencing and Mapping, 1, 375-388 (1991).

[3] Livshits, M.A., Florentiev, V.L., and Mirzabekov, A.D. J. Biomol. Struct. Dynam., 11, 783-795 (1994).

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.