Sanger Sequencing Method

Overview

• Sanger sequencing is a method for determining the nucleotide sequence of DNA.
• It is based on the use of dideoxynucleotides in a DNA polymerization reaction.

Nucleotide Basics

• Nucleotide components:
  • Nitrogenous Base (A, T, G, C)
  • Sugar Residue
  • Phosphate Group
• Nucleoside: Lacks the phosphate group.
• Deoxynucleotide: Lacks the hydroxyl group at the 2' end of the sugar.
  • Used by DNA polymerases as substrate.
  • 3' OH group is crucial for DNA polymerization.

Principle of Sanger Sequencing

• Developed by Frederick Sanger in 1977.
• If the 3' OH group is absent, nucleotide addition stops, terminating the DNA chain.
• Dideoxynucleotide (ddNTP): Missing the 3' OH group (stops the reaction).

Classical Sanger Sequencing (1980s)

• Template DNA is divided into four tubes.
  • Each tube has a primer and all four dNTPs (A, T, G, C).
  • DNA polymerase enzyme is used.
• Each tube contains a low concentration of one radiolabeled dideoxynucleotide:
  • Tube 1: ddATP
  • Tube 2: ddTTP
  • Tube 3: ddGTP
  • Tube 4: ddCTP
• DNA polymerase adds dNTPs; if ddNTP is added, reaction stops.
• Chain termination results in fragments of varying lengths.
• Fragment lengths determined by polyacrylamide gel electrophoresis and autoradiography.
• Sequence deduced from autoradiogram.

Modern Sanger Sequencing

• Each ddNTP is attached to a fluorescent dye (e.g., yellow, green, blue, red).
• Reactions can be done in a single tube due to unique fluorescence of each ddNTP.
• Chain-terminated fragments separated by polyacrylamide gel electrophoresis.
• Advanced methods use capillary electrophoresis for separation.
• Detection achieved with charge-coupled device (CCD) for fluorescent signals.

Key Takeaways

• Sanger sequencing relies on chain termination by ddNTPs.
• Modern methods use fluorescent dyes for easier detection and analysis.
• The method has evolved from using multiple tubes to a single-tube format with improved detection techniques.