New DNA sequencing methods

Sekvenciranje DNA je metoda koja omogućava određivanje redoslijeda nukleotida u DNA molekuli. Metode se dijele na tri generacije, koje se razlikuju po brzini, preciznosti, propusnosti, veličini fragmenata, te načinu njihove detekcije i analize. 1977. godine razvijena je Sangerova metoda, temeljena na sekvenciranju DNA fragmenata pomoću prethodno obilježenih dideoksinukleotid trifosfata koji sprječavaju povezivanje daljnjih nukleotida i time prekidaju sintezu lanca DNA. Paralelno sa Sangerovim otkrićem, 1977. godine Maxam i Gilbert razvijaju metodu temeljenu na nasumičnom cijepanju fragmenata DNA na različitim bazama s obzirom na korišteni agens (hidrazin, dimetil sulfid i dr.). Razvojem tehnologije dolazi do pojave metoda druge generacije kao što je pirosekvenciranje u kojem prilikom polimerizacije DNA dolazi do otpuštanja pirofosfata i emitiranja fotona koji služe za detekciju fragmenata. Pojavom detekcije u stvarnom vremenu otvorene su nove mogućnosti sekvenciranja DNA, poput monomolekulskog sekvenciranja (SMRT), te sekvenciranje pomoću nanopora. Sukladno razvoju novih metoda, otkrivene su nove tehnike sekvenciranja poput 454 pirosekvenciranja, Illumina, SOLiD, te HeliScopea. Korištenje metoda sekvenciranja DNA omogućava napredak brojnih prirodnih znanosti, poput genetike, biomedicine i biokemije, ali i napredak u proučavanju raznih bolesti, kao i u razvoju novih lijekova.DNA sequencing is a method that allows determination of nucleotide order in DNA molecule. Methods are divided into three generations, which differ in speed, precision, throughput, fragment size, and detection and analysis. In 1977, Sanger et al. developed a method based on DNA sequencing by pre-labelled dideoxynucleotide triphosphates, which prevent incorporation of further nucleotides and interrupt synthesis of DNA chain. In 1977, Maxam and Gilbert developed another method based on random cleavage of fragments depending on the used agents (hydrazine, dimethyl sulfide, etc.). Advancement of technology led to the development of the second generation methods, such as pyrosequencing in which DNA polymerization results in the release of pyrophosphates and emission of photons, used for the detection of fragments. Real-time detection opens new possibilities for DNA sequencing, such as single molecule real time sequencing (SMRT) and nanopore sequencing. In accordance with the development of new methods, new sequencing techniques, such as 454 pyrosequencing, Illumina, SOLiD and HeliScope, are discovered. The use of DNA sequencing allows advancement of numerous natural sciences, such as genetics, biomedicine and biochemistry, as well as a progress in studying of various diseases and development of novel drugs