The combination of Gilbert/Maxam chemical sequencing and the dideoxynucleotide chain termination approach facilitates the construction of species specific PCR-primers based on diagnostic RAPD bands.
The randomly amplified polymorphic DNA technique (RAPD) is a modification of PCR that uses short, arbitrarily 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 oligonucleotide primers were synthesized. The value of this approach was demonstrated for the molecular diagnosis of the important rape seed (Brassica napus) pathogen Leptosphaeria maculans.
Voigt
K
K
Institut für Mikrobiologie, Friedrich-Schiller-Universität Jena, Germany.
Wöstemeyer
J
J
D001203:Ascomycota / Q000235:genetics; D001483:Base Sequence; D001937:Brassica / Q000382:microbiology; D017931:DNA Primers / Q000138:chemical synthesis* / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D019105:Random Amplified Polymorphic DNA Technique; D017422:Sequence Analysis, DNA / Q000379:methods*; D013045:Species Specificity
D017931:DNA Primers; D004271:DNA, Fungal