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Sequencing 5. Which reagent don’t you have? DNA polymerase Phosphodiester bond Hydrogen bond 6. Each cycle of the sequencing reaction has distinct three steps, each performed at a different temperature: First Temperature: 96 degrees Second Temperature: 50 degrees Third Temperature: 7. 60 degrees Action: Denature the DNA Action: Attach primers Action: Binding of enzymes to the DNA What causes the DNA to denature in a sequencing reaction? The heat makes DNA’s molecules vibrate faster than they would at lower temperatures. This heat-induced movement causes the two strands of DNA to separate. What type of bonds form between the complementary bases? Hydrogen bonds. Circle the difference(s) between the structures. Direction of DNA synthesis A deoxynucleotide triphosphate (dNTP) A dideoxynucleotide triphosphate (ddNTP) 1 Why do you think a ddNTP would be unable to form a phosphodiester bond? ddNTPs are missing a hydroxyl (-OH) group on the sugar that plays a major role in the formation of phosphodiester bonds. 14. Dideoxynucleotide Color ddG yellow/black ddA green ddT red ddC blue 0 1 2 3 4 5 PEAK___________________________________________________________ NORMAL________________________________________________________ blue 0 1 red black green 3 4 2 blue 5 15. Based upon your sequencing data above, what is the sequence? CTGAC 16. If your template strand is 10 nucleotides long, how long should your sequence be? Five nucleotides. The first five letters of the template correspond to the primer. 17. Why is your final sequence different from your original template sequence? It is the complement of the template sequence. 2