1. Sequencing by the Sanger Dideoxynucleotide Chain Termination Method 1. Prepare replication template denature, add synthetic primer, promote annealing TAGGCGA GATCTG 5’ 3’ CTAGAC 5’ 3’ TAGGCGA GATCTG 5’3’ unknown sequence DNA template known sequence 5’3’ 2. Add components for in vitro replication to each tube, add: - reaction buffer (salts, pH, etc.) - 4 dNTP precursors (1 radioactively labeled) - DNA polymerase ddCTP ddTTP ddGTP ddATP tube with large number of annealed DNA and primer complexes from step 1 distribute into 4 separate tubes 1 dideoxynucleotide ddATP H

2. Sequencing by the Sanger Dideoxynucleotide Chain Termination Method, continued 4. Electrophoresis and visualization of replication products TAGGCGA GATCTG 5’ 3’ CTAGAC 5’ 3’ 3. Replication reactions CTAGAC 5’ CTAGAC 5’ CTAGAC 5’ CTAGAC 5’ { { { { ddCTP ddTTP ddGTP ddATP ddC rxn ddT rxn ddG rxn ddA rxn + - T G C C T A C 5’ 3’ A C G G A T G 5’ 3’

4. Automated DNA Sequencing Use 4 ddNTPs, each labeled with different fluorochrome, in single reaction. Individual products are differentiable by size and attached fluorochrome. As bands migrate during electrophoresis, a detector senses the different fluorochromes and feeds the information to a computer for data analysis.