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Synthesis and enzymatic testing of reversible terminators for SBS,Used
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Based on the stateoftheart DNA sequencing method Sanger sequencing the major aim of future research is to develop a costeffective and gel electrophoresisfree sequencing method. These new sequencing technologies such as the socalled sequencingbysynthesis method (SBS) should be able to detect DNA mutations faster and more accurate than the currently available sequencing technique. The function of a reversible terminator used for SBS is as follows: 3?modified, dyelabeled nucleotides are incorporated by the polymerase complementary to the DNAtemplate (as already known from Sanger sequencing) stopping the DNAsynthesis immediately. After detection of the fluorescent signal, the reversible terminator has to be cleavable in such a way (here: the 3?modification) that the DNAsynthesis can continue (i. e. ?reversible? termination). Thus SBS enables a stepbystep chain elongation with a direct readout of the sequence via fluorescent signals. This PhD thesis covers the synthesis of 3?modified nucleosides as key compounds for these terminators and the evaluation of suitable 3?modification cleavage conditions.
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