By Joseph R. Lakowicz

ISBN-10: 0306473879

ISBN-13: 9780306473876

ISBN-10: 0306479478

ISBN-13: 9780306479472

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Additional info for Topics in Fluorescence Spectroscopy Vol. 7: DNA Technology

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The appropriate acceptor was conjugated to the primary amine group off the modified base following cleavage from the solid support via a succinimidyl ester functional group. The spacer distance between the donor and acceptor was selected by positioning T* within the M13 (-40) primer sequence during solid phase synthesis. The naming of these ET primers followed the convention, donor-spacer (bp)-acceptor. In the case of the FAM and JOE ET primers, a 10 base spacer was used and for TAMRA and ROX, a 3 base spacer was selected.

As can be seen from this equation, the resolution can be improved by increasing the difference in the mobility of the two bands (increase selectivity, gel property) or increasing the plate numbers (narrower bands). 75, two bands are baseline resolved. For DNA sequencing, the accuracy in the base call depends intimately on the resolution obtained during gel fractionation. Common polymers used for DNA sequencing are linear or crosslinked polyacry lamides or polyethylene oxides, both of which possess the appropriate pore size for sorting single-stranded DNAs.

Dye Primer/Terminator Chemistry and Fluorescence Detection Formats In most sequencing applications, dye-labeled primers are used for accumulating sequencing data using automated instruments. This stems from the fact that dye-labeled primers are typically less expensive to use compared to their dye- DNA Sequencing Using Fluorescence Detection 41 labeled terminator counterparts. Also, in most applications, small pieces of DNA (1–2 kbp in length) are cloned into bacterial vectors for propagation (to increase copy number), such as M13s, which have a known sequence and serve as ideal priming sites.

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Topics in Fluorescence Spectroscopy Vol. 7: DNA Technology by Joseph R. Lakowicz


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