OPTIMIZATION OF SPECTROSCOPIC AND ELECTROPHORETIC PROPERTIES OF ENERGY-TRANSFER PRIMERS

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 T 7 priming sequences), and (iv) the dyes chosen as the donor (6-carboxy fluorescein, F; or boxypentyl)-3'-ethyl-5,5'-dimethyloxacarbocyanine, C) and acceptor (F; 5 & 6-carboxyrhodamine-110, R-110; 6-carboxyrhodam ine-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. Th ese 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 do nor and the acceptor. The primer set C4R(110), C4G, C4T, and C4R provi des acceptor emissions of high spectral purity with donor:acceptor emi ssion 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 e xtension DNA fragments. The C4R(110), C4G, C4T, and C4R set is valuabl e for diagnostic applications where minimization of crosstalk between different labels is of particular importance. The set C10R(110), C10G, C10T, and C10R, which uses only rhodamine dyes as accepters, shows si gnificantly improved matching in the electrophoretic mobilities of sin gle-base extension DNA fragments over the previously described set C10 F, C10G, C10T, and C10R and is the best available for sequencing. (C) 1997 Academic Press.