Sanger Sequencing

1) Get Some DNA To Sequence *le pick… *le smash… *le precipitate… *le DNA in le solution

2) Add a Primer That Binds To Area You Want to Sequence *le DNA in le solution * le primer Base pair with gene to sequence 3’ end for adding nucleotides

3) Make Many Copies of the Gene *le DNA + le Primers *le DNA Polymerase *le free nucleotides (dNTPS) Heating Cycle: 1)94 o C to break H-bonds 2)65 o C Primers anneal to ssDNA 3)72 o C DNA Polymerase copies genes using dNTPS 4)Repeat Many Times

How Heating Cycle Works Heating Cycle: 1)94 o C to break H-bonds 2)65 o C Primers anneal to ssDNA 3)72 o C DNA Polymerase copies genes using dNTPS 4)Repeat Many Times *le DNA *le H-bonds

How Heating Cycle Works Heating Cycle: 1)94 o C to break H-bonds 2)65 o C Primers anneal to ssDNA 3)72 o C DNA Polymerase copies genes using dNTPS 4)Repeat Many Times

4) Copy Gene Using Doubly Deoxidised, Florescent NTPs *le many copies of gene + primers *le DNA Polymerase *le free nucleotides (dNTPS) Fluorescent dye (A=Red, C= Blue…) Doubly deoxidised (no 3’ oxygen to form next bond)

How ddNTPs Work Heating Cycle: 1)94 o C to break H-bonds 2)65 o C Primers anneal to ssDNA 3)72 o C DNA Polymerase copies genes using dNTPS 4)Repeat Many Times *le DNA *le H-bonds

How Heating Cycle Works Heating Cycle: 1)94 o C to break H-bonds 2)65 o C Primers anneal to ssDNA 3)72 o C DNA Polymerase copies genes using dNTPS 4)Repeat Many Times

5) Repeat Many Times To Get Copies of Different Lengths all Ending in a ddNTP *le messy mess

6) Sort DNA Segments By Size With Gel Electrophoresis *le not messy mess *le agarose gel *le shockie shockie

7) Use A Laser To “Read” Sections and Sequence DNA *le cool laser *le sequence