Bases teoricas de sanger sequencing

1. Sanger
Sequencing

2. How did your samples get here?
2
DNA extraction – spit into DNAgenotek kit
Gene amplification – PCR
Gene detection – gel electrophoresis
Sequencing preparation – ExoSAP-IT digestion
Two reactions per sample containing:
o Amplified DNA
o Forward OR reverse primer
o water
forward
reverse

3. What happens next?
3
Add Polymerase and Nucleotides*
Perform PCR (with a slight modification*)
Purify DNA samples using magnetic beads

4. Sanger sequencing is a modified
PCR reaction
4
Basic components of PCR
o Polymerase
o Deoxynucleotides
o Template DNA
o Primers
Extra components of Sanger
sequencing
o Dideoxynucleotides

5. DNA - deoxyribonucleotides
5

6. DNA synthesis is a chemical reaction
6
5’
3’ H
H
H
H
5’
3’
5’
3’
phospodiester bond

7. Sanger sequencing uses
DIdeoxyribonucleotides
7
H H
H
H
H
5’
3’ H
5’
3’
5’
3’

8. Sanger sequencing is a modified
PCR reaction (chain terminating)
8
3’ ATGCGGATTAGACATTGAC 5’
5’ TACGCCTAATCT*
5’ TACGCCT*
5’ TACGCCTAATCTGT*
Template
Round 1
Round 2
Round 3
5’ T*
Round 4
5’ TACGCCTAATCTGTAACT*
Round 5

9. Sanger sequencing is a modified
PCR reaction
9
Basic components of PCR
o Polymerase
o Deoxynucleotides
o Template DNA
o Primers
Extra components of Sanger
sequencing
o Some dideoxynucleotides
o Four different reactions

10. 10
Four reactions
o A, T, G, C:C*
o T, G, C, A:A*
o C, A, T, G:G*
o G, C, A, T:T*
Sanger sequencing is a modified
PCR reaction

11. 11
Four reactions
o A, T, G, C:C*
o T, G, C, A:A*
o C, A, T, G:G*
o G, C, A, T:T*
Sanger sequencing is a modified
PCR reaction