Date of Award

5-15-2008

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Chemistry

First Advisor

Markus W. Germann - Chair

Second Advisor

W. David Wilson

Third Advisor

Kathryn B. Grant

Abstract

Standard chemical DNA synthesis with isotope labels requires expensive reagents; moreover, a large excess of phosphoramadites (typically 50-100 fold) must be used. We developed a process where enzymatic cyclic solid phase synthesis of DNA allows for more economic reagent use. A DNA template was immobilized on an epoxy-activated solid support. This chemistry was chosen because the formed linkage is inert to high pH conditions. High efficiency of the covalent attachment was observed when the reaction was carried out in MgCl2/CAPS buffer. It was found that Mg2+ enables the reaction to be completed over a period of 14 h, compared to 72 h under standard conditions. DNA synthesis was carried in a cyclic fashion on a support bound DNA using Klenow fragment.

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