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Unformatted text preview: Fluorescent DNA Base Modifications and Substitutes: Multiple Fluorophore Labeling and the DETEQ Concept H ANS-A CHIM W AGENKNECHT University of Regensburg, Institute for Organic Chemistry, Regensburg, Germany There is an increasing need for fluorescent nucleic acid probes that are able to sense genetic variations without the application of enzymes. The incorporation of organic fluorophores either as DNA base modifications or as DNA base substitutions represents a powerful and versatile method for such new fluorescent DNA assays. Multiple labeling of oligonucleotides using several adjacent chromophore-modified DNA bases yields fluorescence enhancement and modulation that are sensitive to single-base mismatches in the complementary oligonucleotide. Charge transfer processes that cause fluorescence quenching are DNA-base mediated and occur over several base pairs distance. Our “DETEQ” setup, consisting of a fluorescence DNA base substitution and the charge acceptor as a second modification two base pairs away, allows the homogeneous detection of single-base mutations simply by fluorescence readout. This could lead to new DNA microarrays which are based on charge transfer processes and can be analyzed by commonly used fluorescence readout techniques. Key words: chromophore; DNA; electron transfer; fluorescence Introduction Within the entire human genome, which comprises approximately 3.2 billion base pairs, individual human beings differ in approximately 0.1% of their nucleotide sequence. 1 , 2 The most common of these three mil- lion genetic differences are variations of single-base pairs (single-nucleotide polymorphisms, SNPs) within an otherwise unchanged genetic context. With respect to pharmacogenomics, it is known that some of these genetic variations are directly linked to diseases as well as to the observation that drugs have significantly dif- ferent effects on different patients. 3–5 Thus, consid- erable efforts are being spent on the development of reliable and sensitive high-throughput DNA assays for the detection of genetic variations in order to elucidate the connection to the observed phenotype changes. 6–11 Especially, time-, labor-, and cost-effective analytical methods are sought after. 6 Developing these meth- ods will be crucial in the future to allow a therapy that is tailored to the genetic makeup of individual pa- tients. Thus, treatments with drugs that are ineffective Address for correspondence: Hans-Achim Wagenknecht, University of Regensburg, Institute for Organic Chemistry, D-93040 Regensburg, Germany. Voice: Int + 49-941-943-4802; fax: Int + 49-941-943-4617. [email protected] or cause severe side effects due to the individual genetic prerequisites may be avoided....
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This note was uploaded on 07/11/2010 for the course SPECTOGRAP 545 taught by Professor Gdf during the Spring '10 term at AIB College of Business.
- Spring '10