Sanger Sequencing. 1) Get Some DNA To Sequence *le pick… *le smash… *le precipitate… *le DNA...

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) 94oC to break H-bonds2) 65oC Primers anneal to ssDNA3) 72oC DNA Polymerase copies genes

using dNTPS4) Repeat Many Times

How Heating Cycle WorksHeating Cycle:

1) 94oC to break H-bonds2) 65oC Primers anneal to ssDNA3) 72oC DNA Polymerase copies

genes using dNTPS4) Repeat Many Times

*le DNA*le H-bonds

How Heating Cycle WorksHeating Cycle:

1) 94oC to break H-bonds2) 65oC Primers anneal to ssDNA3) 72oC DNA Polymerase copies genes

using dNTPS4) 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 WorkHeating Cycle:

1) 94oC to break H-bonds2) 65oC Primers anneal to ssDNA3) 72oC DNA Polymerase copies

genes using dNTPS4) Repeat Many Times

*le DNA*le H-bonds

How Heating Cycle WorksHeating Cycle:

1) 94oC to break H-bonds2) 65oC Primers anneal to ssDNA3) 72oC DNA Polymerase copies genes

using dNTPS4) 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