Understanding Sanger Sequencing: A Step-by-Step Guide

Explore the detailed procedure of Sanger sequencing, from sample collection to DNA extraction, gene amplification, and sequencing preparation. Learn about the components involved, such as polymerase, nucleotides, and dideoxynucleotides, to achieve accurate DNA sequencing results. Follow the chain-terminating reaction and the incorporation of di-deoxyribonucleotides to decipher DNA sequences effectively.

• Sanger Sequencing
• DNA Extraction
• Gene Amplification
• Polymerase Chain Reaction
• Dideoxynucleotides

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Uploaded on Jul 17, 2024 | 3 Views

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1. Sanger Sequencing

2. How did your samples get here? 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 2

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

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

5. DNA - deoxyribonucleotides 5

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

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

8. Sanger sequencing is a modified PCR reaction (chain terminating) Template 3 ATGCGGATTAGACATTGAC5 5 TACGCCTAATCT* 5 TACGCCT* 5 TACGCCTAATCTGT* 5 T* 5 TACGCCTAATCTGTAACT* Round 1 Round 2 Round 3 Round 4 Round 5 8

9. Sanger sequencing is a modified PCR reaction 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 9

10. Sanger sequencing is a modified PCR reaction 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* 10

11. Sanger sequencing is a modified PCR reaction 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* 11