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Molecular Genetic Advances in the 80s - Coggle Diagram

- - - - T. aquaticus lives in hot springs and hydrothermal vent
  - - - Annealing
        
        Extension
        
        Raise the temperatures (72 C) so Taq polymerase extends the primers, synthesizing new strands of DNA
        
        Cool (55 - 65 C) the reaction so the primers can bind to the ends of the selected section of DNA
      - Heat the reaction strongly (96 C) to denature the DNA strands
  - - - Single strand binding proteins stabilize each half of the DNA
        
        The leading strand is continually synthesized in the 5' to 3' direction by polymerase and is read by polymerase in the 5' to 3' direction
        
        The lagging strand is synthesized in chunks called Okazaki fragments
        
        The primer is replaced and Ligase joins the Okazaki fragments
- - - - Size separation by Gel Electrophoresis
        
        Gel analysis and determination of DNA sequence
        
        by reading the gel bands from smallest to largest, we can determine the 5’ to 3’ sequence of the original DNA strand.
        
        IN MANUAL | the user reads all four lanes of the gel at once, moving bottom to top, using the lane to determine the identity of the terminal ddNTP for each band. For example, if the bottom band is found in the column corresponding to ddGTP, then the smallest PCR fragment terminates with ddGTP, and the first nucleotide from the 5’ end of the original sequence has a guanine (G) base
        
        IN AUTOMATED | A computer reads gel using the florescent labels
        
        oligonucleotides are separated by size via gel electrophoresis. In gel electrophoresis, DNA samples are loaded into one end of a gel matrix, and an electric current is applied; DNA is negatively charged, so the oligonucleotides will be pulled toward the positive electrode on the opposite side of the gel. Because all DNA fragments have the same charge per unit of mass, the speed at which the oligonucleotides move will be determined only by size.
        
        IN MANUAL | Each of the 4 PCR reactions are ran in separate lanes of the gel
        
        IN AUTOMATED | PCR is run in single well in gel in machine
      - Like standard PCR with the inclusion of modified dNTPs called dideoxyribonucleotides (ddNTPs) into the normal dNTPs
      - IN MANUAL | 4 PCR reactions are set up with only one type of ddNTPs
      - IN AUTOMATED | all ddNTPs are mixed in a single reaction (each of the 4 has a different fluorescent label)