Abstract | Deoksiribonukleinska kiselina je molekula sastavljena od 2 polinukleotidna lanca, međusobno povezana vodikovim vezama, koji se spiralno uvijaju. Svaki je lanac polimer nukleotida, osnovne jedinice koja je sastavljena od jedne dušične baze, šećera deoksiriboze i fosfatne skupine. Lanci su međusobno komplementarni i antiparaleleni, a tri nukleotida predstavljaju osnovnu kodirajuću jedinicu, kodon. Kodon kodira određenu aminokiselinu, one se dalje spajaju u polipeptidni lanac pa u protein te, na taj način, DNK nosi informaciju svih funkcija i osobina organizma. Molekula DNK je kompaktno složena u kromosome čiji je broj vrsno specifičan. Kromosomi su smješteni u staničnoj jezgri i predstavljaju genomsku DNK nekog organizama. Neke stanične strukture imaju vlastitu DNK kao što su mitohondriji, bakterijski plazmidi i biljni koloroplasti. Izolacija DNK je danas neizostavan i rutinski postupak u molekularnoj dijagnostici. Prva izolacija DNK je napravljena 1869. godine od strane švicarskog doktora i biologa, Friedricha Mieschera. Danas su razvijene mnogobrojne metode i njihove varijacije koji se odabiru ovisno o vrsti uzorka s ciljem izolacije što kvalitetnije i veće količine nukleinskih kiselina. Sve te metode imaju neke zajedničke korake; skupljanje stanica, otapanje stanične membrane s raznim deterdžentima i surfaktantima, liza proteina i RNK (po potrebi), odvajanje nepotrebnog debrisa, centrifugiranje i pročišćavanje dobivenih nukleinskih kiselina. Čistoća i koncentracija dobivene DNK se zatim ispituje spektrofotometrijom na raznim valnim duljinama i mjerenjem apsorbancije. Kvantifikacija se još može provesti sa rezanjem specifičnim restrikcijskim enzimima i bojenjem etidijevim bromidom. Izolirana DNK se dalje analizira raznim PCR metodama, RFLP analizama, Southern blotting i dr. Uzorci mogu biti razne vrste tkiva, dlaka, tjelesne tekućine, stolica, klinički dobiveni biopsati, FNA dobivene stanice, krv i serum, histološki preparati, kosti, zubi, nokti, sperma, mlijeko, perje, itd. Metode ekstrakcije se mogu podijeliti na metode organske ekstrakcije, metode izdvajanja na silika gelu, magnetsku separaciju, tehnologiju anionske izmjene te ostale. Primarna važnost izdvajanja DNK je u proučavanju genetskih uzroka bolesti, razvoju dijagnostike i lijekova, kao i samog istraživanja njene građe i funkcije. U životinja DNK se izdvaja i analizira u forenzičke svrhe, radi certificiranja čistokrvnih životinja, utvrđivanje očinstva u leglu, trajnog dokaza identiteta i podrijetla, utvrđivanja stupnja srodnosti prije parenja i sklonosti nekim zdravstvenim problemima. Za forenzičke potrebe, razlikuje se forenzička entomologija i forenzička analiza kralježaka. Analiza DNK kukaca ima za zadatak pomoći odrediti vrijeme, mjesto i način smrti u sudskomedicinskim slučajevima, kao i u slučajevima upitne provedbe mjera dezinsekcije i skladištenja hrane. DNK kralježnjaka, poglavito psa i mačke u sudskomedicinske svrhe, se izdvaja kada je životinja „svjedok“, žrtva ili počinitelj. Prehrambeni proizvodi životinjskog podrijetla, i za ljude i za životinje, podliježu kontroli svog deklariranog sastava. Ovaj će rad obuhvatiti neke od najčešćih metoda izolacije ovisno o podrijetlu uzorka, metode kvantifikacije dobivene DNK i moguće daljnje analize ovisno o metodi izolacije. |
Abstract (english) | Deoxyribonucleic acid is a molecule composed of two polynucleotide chains, interlinked with hydrogen bonds and coiling around each other. Each chain is a polymer of nucleotides, basic monomeric units. One nucleotide is composed of one of possible four nitrogen containing nucleobases, sugar deoxyribose and a phosphate group. Chains are mutually complementary and antiparallel with sequence of three nucleotides representing basic coding unit, codon. One codon stands for certain amino acid and they connect into polypeptide chain and further into protein thus the DNK carries information about all functions and characteristics of organism. DNK molecule is organized into structures called chromosomes whose number is species specific. Chromosomes are placed into cell nucleus and represent genomic DNK of that organism. Some cellular strictures have their own DNK as bacterial plasmids, plant chloroplasts and mitochondria. DNK extraction is, today, routine and indispensable procedure in molecular diagnostics. First successful extraction was done in 1869 by a Swiss physician and biologist, Friedrich Miescher. Today there are many developed methods, and their variations, witch are selected based on a type of sample and with the goal to extract as much amount as possible and best quality too. All this methods have some common steps; collecting cells, lysing of the cell wall with detergents and surfactants, protein lysis and RNK (if not needed), removal of unnecessary debris, centrifugation and purification of acquired nucleic acids. Purity and concentration of given DNK are tested trough spectrophotometry on different wave lengths and measuring absorbance. Quantification can also be measured by cutting sample with restriction enzymes and dying with ethidium bromide. Isolated DNK is further analyzed with many PCR methods, RFLP analysis, Southern blotting, etc. Samples can be all sorts of tissues, hairs, body fluids, stool, clinically acquired bioptates, FNA acquired cells, blood and serum, histological samples, bones, teeth, nails, semen, milk, feathers, etc. Extraction methods can be divided into organic extraction methods, separation on silica gel, magnetic separation, anionic exchange based technology methods and others. Biggest relevance of DNK extraction is to study genetic causes of diseases, development of diagnostics and treatment and to further investigate structure and function of DNK. In animals, DNK is isolated and analyzed for forensic purposes, to certify purebred animals, determining fatherhood in offspring, as a permanent evidence of identity and origin, determining degree of cognation before mating and proneness to certain diseases and conditions. In forensic science, we distinguish between forensic entomology and forensic analysis of vertebrates. Insects DNK analysis is used to help determine time, place and way of death in forensic cases as in cases of questionable implementation of disinsection and food storage measures. Vertebrate DNK, mainly cats and dogs is used in forensic cases when animal can be in role of witness, victim or perpetrator. Food product of animal origin, for use in humans and animals, are subject to control of their declared composition. This graduate thesis will incorporate some of most common methods of DNK extraction depending on the type of sample, methods of quantification of obtained DNK and possibilities of downstream applications depending on method of isolation. |