Isolation of microsatelite markers by FIASCO method | Izolacija mikrosatelitnih markerjev z metodo FIASCO

Inglés. 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.

Esloveno. Metoda FIASCO (Fast Isolation by AFLP of Sequences Containing Repeats) omogoča razmeroma hitro izolacijo mikrosatelitnih markerjev. Primerna je za vse organizme, ne glede na to, kako pogosti so mikrosatelitni lokusi v genomu. Z metodo FIASCO smo izolirali mikrosatelitne markerje iz ribona (Pagellus erythrinus). Za restrikcijo DNK smo uporabili endonukleazo MseI. Razrezano DNK smo ligirali z oligonukleotidnima adapterjema MseIU in MseID. Fragmente DNK z adapterji smo pomnožili s PCR, v kateri smo uporabili degeneriran začetni oligonukleotid MseI-N. Ugotovili smo najugodnejše pogoje za PCR. Pomnožene fragmente DNK smo hibridizirali z biotinilirano sondo (AC)12. Kompleks sonda-DNK smo ujeli na magnetnih kroglicah, prekritih s streptavidinom, in z mikrosateliti obogateno frakcijo pomnožili s PCR. Obogateno mikrosatelitno DNK smo klonirali v plazmid pCR® 2.1-TOPO in z njim transformirali celice Escherichia coli TOP10. Pridobili smo 2436 belih transformant. Nukleotidna zaporedja smo določili 18 transformantam. Ugotovili smo, da 88,8 odstotkov transformant vsebuje dinukleotidne mikrosatelitne ponovitve. Našli smo tudi eno tetranukleotidno ponovitev.