Microsatellite analysis using a two-step procedure for fluorescence labeling of PCR products

A method for fluorescent labeling of PCR products has been developed. This method consists in a two-step procedure in which a first exponential classi cal PCR is followed by a "linear amplification". This second step relies on incorporation of fluorescent dNTP (dUTP or dCTP) in order to label the pro duct on only one strand. The products can be applied without prior purifica tion directly to a gel on a fluorescence-based automated DNA sequencer, for length and allele determination. The reliability of the results equals tho se of the classical P-32 or fluorescent primer labeling methods, and the me thod is definitely less costly Since the interpretation of the results is e asier than with the method consisting in a fluorescent dNTP uptake in both strands in a single PCR, the present strategy should prove useful in mappin g projects requiring analysis of a large number of microsatellites.