11 Mart 2008 Salı
By the early 1970s, Sanger was interested in deoxyribonucleic acid (DNA). DNA sequence studies had not developed because of the immense size of DNA molecules and the lack of suitable enzymes to cleave DNA into smaller pieces. Building on the enzyme copying approach used by the Swiss chemist Charles Weissmann in his studies on bacteriophage RNA, Sanger began using the enzyme DNA polymerase to make new strands of DNA from single-strand templates, introducing radioactive nucleotides into the new DNA. DNA polymerase requires a primer that can bind to a known region of the template strand. Early success was limited by the lack of suitable primers. Sanger and British colleague Alan R. Coulson developed the “plus and minus” method for rapid DNA sequencing. It represented a radical departure from earlier methods in that it did not utilize partial hydrolysis. Instead, it generated a series of DNA molecules of varying lengths that could be separated by using polyacrylamide gel electrophoresis. For both plus and minus systems, DNA was synthesized from templates to generate random sets of DNA molecules from very short to very long. When both plus and minus sets were separated on the same gel, the sequence could be read from either system, one confirming the other. In 1977 Sanger's group used this system to deduce most of the DNA sequence of bacteriophage FX174, the first complete genome to be sequenced.