Generation of Sequence Signatures from DNA Amplification Fingerprints with Mini-Hairpin and Microsatellite Primers

摘要

DNA amplification fingerprinting (DAF) with mini-hairpins harboring arbitrary "core" sequences at their 3' termini were used to fingerprint a variety of templates, including PCR products and whole genomes, to establish genetic relationshipsbehveen plant taxa at the interspecific and intraspecific level, and to identify closely related fungal isolates and plant accessions. No correlation was observed between the sequence of the arbitrary core, the stability of the mini-hairpin structure andDAF efficiency. Mini-hairpin primers with short arbitrary cows and primers complementary to simple sequence repeats present in mi-cmsatellites were also used to generate arbitrary signatures from amplification profiles (ASAP). The ASAP strategy is adual-step amplification procedure that uses at least one primer in each fingerprinting stage. ASAP was able to reproditcibly amplify DAF products (representing about 10-15 kb of sequence) following careful optimization of amplification parameters such asprimer and template concentration. Avoidance of primer sequences partially complementary to DAF product termini was necessary in order to produce distinct fingerprints. This allowed the combinatorial use of oligomers in nucleic acid screening, with numerous ASAP fingerprinting reactions based on a limited number of primer sequences. Mini-hairpin primers and ASAP analysis significantly increased detection of polymorphic DNA, separating closely related bermudagrass (Cynoclon) cultivars and delecting pitlatively linked markers in bulked segregant analysis of the soybean (Glycine max) supernodulation (nitrate-tolerant symbiosis) locus.