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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.

Quantitative Comparison of the Incorporation of Borano- and Thio- Deoxynucleoside Triphosphate Analogs by Klenow Polymerase

Kenneth Porter, Katie Ealey, J. David Briley, Faqing Huang and Barbara Ramsay Shaw
Department of Chemistry, Duke University, Durham, N.C. 27708

A new class of phosphate-substituted nucleotides, the 2'-deoxynucleoside 5'-alpha-borano triphosphates, has been shown to be good substrates for DNA polymerases. These boranophosphates form the basis of a sequencing method wherein DNA is subjected to repeated amplification/denaturation in the presence of both normal and alpha-boranophosphates, and then treated with exonuclease to produce fragments truncated at the nearest boranophosphate nucleotide (see accompanying abstract by Porter, Briley and Shaw).

The kinetic parameters for the incorporation of borano-analogs were determined by steady-state kinetic analysis and compared to the corresponding thio- and normal dNTP analogs. Labeled primer was extended by exonuclease-free Klenow in the presence of various concentrations of the appropriate dNTP or dNTP analog and the percent extension was quantitated and converted to a measurement of the initial reaction velocity. Values for kCat and Km were calculated from a Michaelis-Menten plot by nonlinear regression. The kCat values for both the borano- and the thio- nucleotide analogs were about 40% that of normal dNTPs. However, the Km values for boronated nucleotides (except for T) were less than the Km values of normal dNTPs, while the Km values for thiophosphates were significantly higher than borano and normal dNTPs. The efficiency of incorporation (kcat/Km) for the boranophosphates was approximately 30% that of normal dNTPs, whereas the efficiency of the thiophosphates was approximately 15% that of normal dNTPs. Therefore, the boranophosphates, although less efficient substrates than normal dNTPs, were shown to be twice as efficient as the thiophosphates.