Universal fluorescent labeling (UFL) method for automated microsatellite analysis.
We have devised a novel method for automated microsatellite analysis using "universal" fluorescent labeling. This system is based on polymerase chain reactions driven by sequence-specific primers and a reporter primer labeled with a fluorescent dye at its 5' end. The forward sequence-specific primer is designed with a tag region bearing no homology to any human genomic sequence. Complementary tag sequences act as templates for the 6-carboxyfluorescein-labeled reporter primer, and those products can be analyzed with an autosequencer. The results we achieved with this assay system were consistent with the results of conventional assays using radioisotope-labeled primers, and diagnosis required less time. Furthermore, the fluorescent-labeled reporter primer is "universal" in that it can be used with different sequence-specific primers designed to carry the appropriate tag sequence at their 5'-ends. Our observations suggest that the "universal" fluorescent labeling method is an efficient tool for analyzing sequence variations in human DNA.
Shimizu
Masashi
M
Department of Molecular Biology, Institute of Gerontology, Nippon Medical School, 1-396 Kosugi-cho, Nakahara-ku, Kawasaki 211-8533, Japan.
Kosaka
Natsuki
N
Shimada
Takashi
T
Nagahata
Takemitsu
T
Iwasaki
Hironori
H
Nagai
Hisaki
H
Shiba
Tadayoshi
T
Emi
Mitsuru
M
D017931:DNA Primers; D005452:Fluoresceins; D018895:Microsatellite Repeats; D010761:Phosphorus Radioisotopes; D013194:Staining and Labeling / Q000379:methods*
D017931:DNA Primers; D005452:Fluoresceins; D010761:Phosphorus Radioisotopes; C024098:6-carboxyfluorescein