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DNA Sequencing Biotechnology II Essential Question What technology is used to determine the sequence of DNA? DNA Sequencing Once DNA amplified, its nucleotide sequence determined Today, DNA sequencing is automated Automated system originally based on Dideoxy Chain Termination Method Invented by Frederick Sanger - received Nobel Prize in 1980 Deoxy Chain Termination Method Based on DNA replication Makes use of dideoxyribonucleotides Remember deoxyribose found in DNA– missing an oxygen at 2 carbon Dideoxyribose – missing two oxygens – at 2 and 3 carbon Significance: Oxygen at 3rd Carbon required for DNA strand extension Where free nucleotide attached When dideoxyribose incorporated into DNA molecule, DNA extension stops DNA Sequencing Reaction Mixture Gene to be sequenced denatured and incubated in test tube with • primer complementary to 3’ end of gene, • DNA polymerase, • four deoxyribonucleotides • four dideoxyribonucleotides tagged with radiolabel 32P DNA Sequencing Steps Synthesis of new strand starts at 3’ end of primer and continues until dideoxyribonucleotide is inserted- stops synthesis Separate labeled strands through a polyacrylamide gel Placed on x-ray film Because of florescent tag, different length fragments are revealed on film and read manually Example of DNA sequence Problem with Sanger’s Method Labor intensive Took a long time to sequence a gene that was fairly large in size (1000-2000 bp) Often times mistakes made in interpreting gels Advancement in Technology First major advancement came with the creation of florescent tags for dideoxyribonucleotides rather than radiolabel Each dideoxy base given a different color tag Example Adenine – green Thymine –red Guanine – yellow Cytosine -Blue Florescent label can now be read with laser detector eliminating radiolabel and xray film Advancement in Technology Eventually, the whole process became automated Contain detector systems that detect the florescent dye Class Activity Get into groups and complete the simulation on the dideoxy method initially developed by Sanger