Isolation of microsatelite markers by FIASCO method

Abstract

The FIASCO (Fast Isolation by AFLP of Sequences Containing Repeats) method enables relatively fast isolation of microsatellite markers. It is appropriate for all organisms without regard to how frequent the microsatelite loci in the genome are. By FIASCO method we isolated microsatelite markers in the seabram (Pagellus erythrinus). DNA restriction was performed by MseI endonuclease. Restricted DNA was ligated with adapters MseIU and MseID. DNA fragments were amplified by PCR using degenerate primer MseI-N. We determined optimal conditions for PCR. Amplified DNA fragments were hybridized with biotinilated probe (AC)₁₂. Probe-DNA complex was captured on magnetic beads coated with streptavidin, and an enriched fraction with microsatellites was amplified by PCR. Enriched microsatelite DNA was cloned into plasmid pCR^® 2.1-TOPO and further transformed into Escherichia coli TOP10 cells. Twothousand fourhundred thirthysix white transformants were obtained and 18 of them were sequenced. We determined that 88.8 percentage of transformants containing dinucleotide microsatellite repeats. One tetranucleotide repeat was also found.