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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.

Positional Effects of Nearest-Neighbor Base Pairs and Mismatched Base Pairs In Short DNA Duplexes

Mitchel J. Doktycz, Maxwell Morris[1], K. Bruce Jacobson, Kenneth L. Beattie[3], and Robert S. Foote[2]
Health Sciences Research Division, [1]Math and Computer Sciences Division, and [2]Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-8080.
[3]DNA Technology Laboratory, H.A.R.C., The Woodlands, Texas 77381

The careful characterization of the hybrid stability of short DNA molecules will be necessary for the success of SBH as a sequence diagnostic method. Knowing the conditions to achieve maximal hybrid stability will allow design of protocols to achieve optimal discrimination against mismatched hybrids. Furthermore, knowledge of the hybrid stability will be a guide in selection of sample preparation procedures, in the selection and positioning of the immobilized DNAs and in effective labeling and detection schemes.

Prediction of the duplex stability of molecules of the type to be used in SBH can be based on thermodynamic stability data. Such data have been obtained primarily from experiments on longer DNAs. For shorter oligomers, end effects, which refer to the initiation of melting from the solvent exposed ends, complicate the application of thermodynamic data collected on long DNAs to the types of molecules used in SBH. This effect may be propagated several base pairs in from the end and can contribute to destabilization of the end base pairs and reduced diagnostic accuracy by increasing the relative stability of mismatches. To evaluate this interaction and others which may be peculiar to small DNA molecules, a systematic evaluation of solution hybridizations has been conducted. The molecule sets studied are based on octamers of the sequences 5'NNNTGGAC3' (set 1), 5'GCNNNGAC3' (set 2) and the respective complements 5'GTCCANNN3' and 5'GTCNNNGC3', where N = A, T, G, or C for a total of 256 molecules. Modeling of melting temperatures of the 128 perfectly matching duplex molecules have allowed evaluation of the stability of A-T and G-C base pairs and the 10 nearest neighbor stacking interactions as a function of base pair position. These same molecule sets have also been used for the evaluation of the 8 mismatched base pairs as a function of position.

Concurrent with the optical melting studies is an examination of the hybridization of these octamers to immobilized 8-mers. DNA sequences corresponding to either one half of the set 1 or set 2 oligomers have been synthesized, using photodeprotection, onto a glass surface. Representative, complementary molecules were radioactively labeled and hybridized to determine the hybridization specificity.

(Research sponsored by the Office of Health and Energy Research, United States Department of Energy, under contract DE-AC05-84OR21400 with Martin Marietta Energy Systems, additional support was received from NIH Grant 1 P20 HG00666.)