Fluorescence energy transfer dye-labeled primers for DNA sequencing and analysis

摘要: Abstract Fluorescent dye-labeled DNA primers have been developed that exploit fluorescence energy transfer (ET) to optimize the absorption and emission properties of label. These carry a fluorescein derivative at 5' end as common donor other rhodamine derivatives attached modified thymidine residue within primer sequence acceptors. Adjustment donor-acceptor spacing through placement in allowed generation four primers, all having strong excitation wavelength (488 nm) maxima 525, 555, 580, 605 nm. The ET efficiency these ranges from 65% 97%, they exhibit similar electrophoretic mobilities by gel electrophoresis. With argon-ion laser excitation, sequencing fragments generated with is 2- 6-fold greater than corresponding or labeled single dyes. higher intensity allows one-fourth template typically required when using T7 polymerase. single-stranded M13mp18 template, typical reaction on commercial sequencer provided sequences 99.8% accuracy first 500 bases. should be generally useful development multiplex analysis methods.