title: Improved AFLP protocol using dual-suppression PCR and its application to species with large genomes.
abstract: To improve the amplified fragment length polymorphism assay, dual-suppression PCR was introduced into the preamplification step of the assay. The dual-suppression PCR blocked completely the amplification of fragments with the same sequence (Bsp1407I-Bsp1407I or NlaIII-NlaIII) at both ends and amplified selectively fragments with different adaptor sequences (Bsp1407I-NlaIII) at each end. Two protocols, referred to as A and B, were established for species with medium- and large-sized genomes, respectively. Both protocols incorporated the dual-suppression PCR. Protocol A resulted in high-quality electrophoretic profiles for black cottonwood and rice, which have medium-sized genomes. In protocol B, an intensely selective PCR step was added to protocol A. Protocol B yielded profiles for Japanese black pine and Japanese cedar that were improved significantly relative to protocol A: the number of strong peaks increased and that of low peaks decreased. Japanese black pine and Japanese cedar have large genomes. The optimal profiles were generated with a total of eight or nine selective nucleotides.
authors.affiliation: Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka 812-8581, Japan.
authors.forename: Lanhua
authors.initials: L
authors.lastname: Guan
authors.forename: Susumu
authors.initials: S
authors.lastname: Shiraishi
mesh_terms: D054458:Amplified Fragment Length Polymorphism Analysis / Q000379:methods*; D032485:Cryptomeria / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D018745:Genome, Plant / Q000235:genetics*; D012275:Oryza / Q000235:genetics; D028223:Pinus / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D032107:Populus / Q000235:genetics; D013045:Species Specificity
chemical_list: D017931:DNA Primers