Introduction to the Workshop
URLs Provided by Attendees

Abstracts

Mapping

Informatics

Sequencing

Instrumentation

Ethical, Legal, and Social Issues

Infrastructure

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.

Preparation of Oligonucleotide Arrays for Hybridization Studies

Michael C. Pirrung, Steven W. Shuey, David C. Lever, Lara Fallon, J.-C. Bradley, and William P. Hawe
P. M. Gross Chemical Laboratory, Department of Chemistry, Box 90346, Duke University, Durham, NC 27708.

This project is aimed at developing reliable, high-quality chemical synthetic methods to prepare high-density arrays containing thousands of short DNA sequences. Such arrays can be used to sequence DNA by hybridization using principles enumerated by several groups.[1] Arrays consisting of complete sets of DNA of a given length can be prepared by the novel technique of light-directed synthesis,[2] and photoremovable groups are key to this method. We have developed a superior new photoremovable group for light-directed DNA synthesis that gives a byproduct that is chemically inert and readily measured by optical and fluorescence methods, permitting the yield in each of the photochemical deprotection steps to be verified. The 3',5'dimethoxybenzoin (DMB) protecting group has been developed for the phosphotriester method of DNA synthesis.[3] It has been used to prepare by solution synthesis several trinucleotides that include sequences containing 5MeC and I. The unnatural nucleotides are intended to 1) increase the melting temperature of hybrids and 2) increase the discrimination (deltaTm) between one-base mismatches end at the ends of the oligomers (fraying) and perfect hybrids by flanking the reading sequences with a "universal" base that will non-specifically increase base stacking and hydrogen bonding and thereby the Tm. DNA prepared with this method is tethered to the solid phase through its 5' end. The 3',5'-dimethoxybenzoincarbonate photoremovable protecting group has been developed for the phosphoramidite method of DNA synthesis. It has been used to prepare a mixed-sequence decanucleotide by solid-phase synthesis. The purity and sequence of this compound was verified by removal from the support followed HPLC analysis, end-labeling and PAGE analysis, and snake venom phosphodiesterase-catalyzed degradation to mononucleotides followed by HPLC analysis. DNA prepared with this method is tethered to the solid phase through its 3' end. Finally, the 3',5'-dimethoxybenzoincarbonate method has been applied to phosphoramidite synthesis of DNA tethered to the solid phase through its 3' end, permitting the use of arrays in enzymatic transformations such as primer extensions and ligations.

References

[1.] Bains, W.; Smith, G. C. J. Theor. Biol. 1988, 135, 303-307. Drmanac, R.; Labat, I.; Brukner, I.; Crkvenjakov, R. Genomics 1989, 4, 114-128. Khrapko, K. R.; Lysov, Y. P.; Khorlyn, A. A.; Shick, V. V.; Florent'ev, V. L.; Mirzabekov, A. D. FEBS Lett. 1989, 256, 118-122. Lysov, Y. P.; Florent'ev, V. L.; Khorlyn, A. A.; Khrapko, K. R.; Shick, V. V.; Mirzabekov, A. D. Dokl. Akad. Nauk. SSSR 1989, 303, 1508-11.
[2.] Fodor, S.P.A.; Read, J.L.; Pirrung, M.C.; Stryer, L.; Liu, A.T.; Solas, D. Science 1991, 251, 767.
[3.] Pirrung, M. C.; Shuey, S. W. J. Org. Chem. 1994, 59, 0000.