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Sanger Sequencing. 1) Get Some DNA To Sequence . *le pick…. *le smash…. *le DNA in le solution. *le precipitate…. 2) Add a Primer That Binds To Area You Want to Sequence . * l e primer. *le DNA in le solution. Base pair with gene to sequence. 3’ end for adding nucleotides. - PowerPoint PPT Presentation
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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