Izolacija mikrosatelitnih markerjev z metodo FIASCO = Isolation of microsatellite markers by FIASCO method
Lavrič, Miha ; Ramšak, Andreja
Metoda FIASCO (Fast Isolation bz 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. Pomnoene 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 pomnoili s PCR. Obogateno mikrosatelitno DNK smo klonirali v plazmid pCRr 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.
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