Electrophoresis-based sequencing system

Several proprietary fluorescence dye-based detection systems that characterize and quantify probe-bound nucleotide sequences have been developed and commercialized in recent years. For example, general and nonspecific DNA dyes can be used that bind with any double-stranded DNA (i.e., the probe-strand complex or otherwise) and are useful in gel electrophoresis. Much more sophisticated systems rely on oligonucleotide probes that incorporate fluorescent dyes that illuminate when a match between complementary strands are made. Included among the latter are TaqMan , molecular beacons, and Scorpion probes. 

An automatic sequencing instrument has been developed that uses the chain-terminator method. To avoid the use of radioactive labels, a different color fluorescent dye is attached to the primer in each of the four reactions used to synthesize the DNA fragments. The mixture of fragments from all four reactions is then analyzed using electrophoresis in a single lane. A fluorescent spot appears for each polynucleotide of increasing size. The 3 -terminal base for each spot can be determined by the color of the fluorescence. The detection system is computer controlled, and the acquisition of data is automated. A schematic representation... 

As for all synthetic products to be tested in biological systems, a careful analytical characterization of peptide libraries is crucial in order to confirm their identity and establish their quality. Compared to individual peptides, however, the analysis of peptide libraries is complicated due to the fact that the peptides are either bound to a solid support or arranged in highly complex mixtures. This poses certain restrictions on which analytical methods can be used to characterize combinatorial libraries. For example, analytical methods that are based on the separation of product components, such as high performance liquid chromatography (HPLC) and capillary electrophoresis (CE), are only of limited use for the analysis of peptide libraries, in particular of those made up of complex nnixtures (>100 peptides per mixture). The analytical methods beneficially applicable to peptide libraries include amino acid analysis, mass spectrometry, and sequencing. 

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