Autor: dipl. biolog – master Vladimir Vukić
E-mail: vladimirvukic@gmail.com
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Kao markeri se mogu koristiti genske, intergenske ili slučajne sekvence DNK. Odabir markera zavisi od cilja rada, uzorka koji se ispituje i od tehničkih mogućnosti laboratorije. Markeri koji se koriste za identifikaciju vrsta razlikuju se u zavisnosti od biologije vrste i njihove evolutivne udaljenosti. Odabir markera zavisi i od nivoa genetičkih informacija koje posedujemo za date vrste. Potrebno je utvrditi markere specifične za vrstu i upotrebom odgovarajućih prajmera proveriti prisutnost u uzorku.
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Genske sekvence
Genske sekvence koje se koriste kao markeri mogu biti poreklom iz nuklearnog, mitohondijskog i hloroplastnog (ukoliko dati organizam poseduje hloroplaste) genoma. Genske sekvence evoluiraju različitim brzinama, npr. geni za ribozomalne RNK i proteine evoluiraju veoma sporo i pogodni su za filogenetska istraživanja na različitim nivoima. U prehrambenoj industriji se najčešće koriste:
• mitohondrijalni geni za citohrom b, 12 S rRNK, 16 S rRNK, citohrom oksidazu i D petlja
• nuklearni geni za 18 S rRNK, 5S rRNK, α-aktin β-kazein (za mlečne proizvode)
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Intergenske sekvence
– Mikrosateliti (SSR – Simple Sequence Repeats)
Mikrosateliti su segmenti DNK sačinjeni od nekoliko uzastopno ponovljivih motiva koji se sastoje od 1-10 baznih parova. Zastupljeni su ravnomero u genomu svih eukariota i visoko polimorfni (Sl. 8). Sreću se još i nazivi Sequence-Tagged Microsatellite Sites (STMS)
Simple Sequence Repeat Polymorphisms (SSRPs).
Regioni susedni mikrosatelitima mogu biti jedinstveni, što omugućuje sintezu prajmera specifičnih za dati lokus. Nasleđuju se kodominantno, što znači da se mogu detektovatii heterozigoti. Date osobine omogućuju da se primenom standarda identifikuje pripadnost datoj vrsti.
Razlike u mikrosatelitskim sekvencama
– RAPD – Random Amlification of Polymorphic DNA
Metoda se zasniva na korišćenju slučajnih oligonukleotidnih prajmera (10 baznih parova) za PCR umnožavanje. Razlike u sekvencama DNK između vrsta dovode do različitog umnožavanja DNK (Sl. 9). Produkti se razdvajaju na agaroznom gelu uz prisustvo etidijum bromida i posmatraju pod UV svetlom. Upoređivanjem sa poznatim uzorkom se može proveriti pripadnost datoj vrsti.
Umnožavanje RAPD tehnikom
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