An economic method for the fluorescent labeling of PCR fragments

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Abstract

A poor man's approach to genotyping for research and high-throughput diagnostics.

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Fluorescence detection in automated DNA sequence analysis.

Lloyd Smith, Jane Z. Sanders, Robert Kaiser … (1986)

We have developed a method for the partial automation of DNA sequence analysis. Fluorescence detection of the DNA fragments is accomplished by means of a fluorophore covalently attached to the oligonucleotide primer used in enzymatic DNA sequence analysis. A different coloured fluorophore is used for each of the reactions specific for the bases A, C, G and T. The reaction mixtures are combined and co-electrophoresed down a single polyacrylamide gel tube, the separated fluorescent bands of DNA are detected near the bottom of the tube, and the sequence information is acquired directly by computer.

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Energy transfer primers: A new fluorescence labeling paradigm for DNA sequencing and analysis

Richard Mathies, Alexander Glazer, Jingyue Ju (1996)

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Optimization of spectroscopic and electrophoretic properties of energy transfer primers.

Chad Glazer, R Mathies, Karen Hung (1997)

We have synthesized and characterized the spectroscopic properties of 56 energy transfer (ET) fluorescent dye-labeled primers differing in (i) the spacing between the donor and acceptor, (ii) the nature of the spacer (either oligonucleotide or polydideoxyribose phosphate), (iii) the primer sequence (M13 (-40), M13 (-21), M13 reverse, SP6, T3, and T7 priming sequences), and (iv) the dyes chosen as the donor (6-carboxyfluorescein, F; or 3-(epsilon-carboxypentyl)-3'-ethyl-5,5'-dimethyloxacarbocyanine, C) and acceptor (F; 5 & 6-carboxyrhodamine-110, R110; 6-carboxyrhodamine-6G, G; N,N,N',N'-tetramethyl-6-carboxyrhodamine, T; and 6-carboxy-X-rhodamine, R) chromophores. This study led to the development of two significantly improved ET primer sets for multiple-color analyses. These primers are named using the convention D-N-A, where D is the donor, A is the acceptor, and N is the number of nucleotides between the donor and the acceptor. The primer set C4R110, C4G, C4T, and C4R provides acceptor emissions of high spectral purity with donor:acceptor emission ratios of < 0.002 for C4G, < 0.004 for C4T, and < 0.005 for C4R and excellent matching in the electrophoretic mobilities of single-base extension DNA fragments. The C4R110, C4G, C4T, and C4R set is valuable for diagnostic applications where minimization of crosstalk between different labels is of particular importance. The set C10R110, C10G, C10T, and C10R, which uses only rhodamine dyes as acceptors, shows significantly improved matching in the electrophoretic mobilities of single-base extension DNA fragments over the previously described set C10F, C10G, C10T, and C10R and is the best available for sequencing.