Microbiol. Res. (1995) 150, 373 - 377 Technical Report Microbiological Research © Gustav Fischer Verlag Jena The combination of Gilbert/Maxam chemical sequencing and the dideoxynucleotide chain termination approach facilitates the construction of species specific peR -primers based on diagnostic RAPD bands Kerstin Voigt, Johannes Wostemeyer Institut fiir Mikrobiologie, Lehrstuhl flir Allgemeine Mikrobiologie und Mikrobengenetik, Friedrich-Schiller-Universitat Jena, Neugasse 24, D-07743 Jena, Germany Accepted: August 29, 1995 Abstract The randomly amplified polymorphic DNA technique (RAPD) is a modification of peR that uses short, arbitra- rily generated single primers to amplify genomic DNA. Amplified DNA-fragments are often polymorphic and can be used as individual, population- or species-specific markers. Because the RAPD technique requires a very high degree of reproducibility at the instrumentation level and with regard to buffer conditions, we propose to synthesize highly specific conventional PCR primers, the sequence of which is based on the primary diagnostic RAPD bands. In this communication we present a fast and convenient experimental strategy for converting the non-stringent RAPD conditions with their low annealing temperatures to stringent PCR conditions. Diagnostic RAPD bands were sequenced by a combination of chemical (Gilbert/ Maxam) and chain termination (Sanger) techniques. Based on this sequence information, highly specific oligonucleo- tide primers were synthesized. The value of this approach was demonstrated for the molecular diagnosis of the important rape seed (Brassica napus) pathogen Lepto sphaeria maculans. Key words:RAPD-PCR - Gilbert/Maxam sequencing - molecular diagnosis - plant pathogen - Leptosphaeria maculans - Brassica napus Corresponding author: Kerstin Voigt Introduction The 'Random amplified polymorphic DNA' (RAPD) technique is a widely used approach for obtaining physical genetic markers' (Welsh and McClelland 1990; Williams et al. 1990). RAPD is comparable with RFLP, but works faster, needs fewer experimental steps and does not depend on cloned hybridization probes or on sequence infor- mation about the amplified regions. The RAPD- method utilizes but a single primer, typically with a length of ten nucleotides. It has also proven valuable to use RAPD bands as hybridization probes in studies of population genetics, thus exploiting the ease of obtaining specific RAPD markers for conven- tional RFLP analysis. The combination of these methods often allows the unequivocal distinction of organisms at various taxonomic levels. Differences at high taxonomic levels, or between genera, even between individual genotypes within a single species can be revealed (Williams et al. 1990; CrO\yhurst et al. 1991; Schafer and Wostemeyer 1992; Lafranco et al. 1993; Burmester and Wostemeyer 1994;:Brikun et al. 1994). Although RAPD opens the possibility to characterize largely unknown genomes at the mole- cular level without the need for sequence information (Wostemeyer et al. 1992), the method has its dis- advantages. The successful and reproducible perfor- mance of RAPD analyses is experimentally not Microbiol. Res. 150 (1995) 4 373