Sanger Sequencing

Introduction

• Frederick Sanger: Developed the method in 1977 for sequencing DNA using chain-terminating inhibitors.
• Objective: Determine the sequence of nucleotides in DNA.
• Key Component: Chain-terminating inhibitors known as DDNTPs.

DNA Structure and Components

• DNA: Composed of four nucleotides (DNTPs - deoxyribonucleoside triphosphates).
• Components of DNTP:
  • Deoxyribose sugar (one less oxygen than ribose)
  • Nitrogenous base (guanine, cytosine, thymine, or adenine)
  • Triphosphate group
• DDNTPs: Dideoxyribonucleoside triphosphates, lacking two oxygens, terminate DNA chain extension.

DNA Polymerase and Chain Termination

• DNA Polymerase Role: Adds new bases to a growing DNA strand, catalyzing the reaction between the DNTP's phosphate and ribose oxygen.
• Chain Termination: Occurs when a DDNTP is added, preventing further DNTP addition.
• Orientation: DNA extends from 3’ end due to oxygen availability; sequence read from 5’ to 3’ direction.

Original Sanger Sequencing Method

• Components Needed: Primer, DNA polymerase, DNTPs, DNA template, DDNTPs.
• Process Overview:
  • Heat DNA to separate strands.
  • Cool to bind sequencing primers.
  • Add DNA polymerase, dNTPs, and a specific DDNTP.
  • Random incorporation of DDNTP causes chain termination.
  • Resulting fragments differ in length, allowing sequence determination.
• Detection: Gel electrophoresis used to separate fragments; radioactive tags or dyes visualize them.

Automation and Advances

• Challenges: Original method was labor-intensive and slow; advancements aimed to streamline and automate.
• AB370A Instrument:
  • Introduced by Applied Biosystems in 1987.
  • Fluorescent dyes replaced radioactive dyes, safer and more efficient.
  • Automated base calling via computer.
• Human Genome Project (1990): Aims to sequence entire human genome; Sanger sequencing played a crucial role.

Innovations in Sanger Sequencing

• PCR and TAC Polymerase: Enabled cycle sequencing, improving signal strength and reducing DNA requirement.
• Capillary Electrophoresis:
  • Led to instruments like ABI Prism 310 and 3700.
  • Faster, more efficient sequencing processes.
• Big Dye Terminators (1997): Allowed sequencing reactions in a single tube with even peak heights.

Impact of ABI Prism 3700

• Celera vs. Human Genome Project: Race to sequence the human genome, both used ABI Prism 3700.
• Efficiency: Significant reduction in sequencing cost and time.
• Output: Enabled sequencing of thousands of samples daily with minimal manual intervention.

Comparison with Next Generation Sequencing (NGS)

• Accuracy: Sanger - 99.9%, NGS - 99 to 99.9%.
• Cost Effectiveness: Sanger more cost-effective for <20 samples; NGS for larger datasets.
• Sensitivity: NGS more sensitive in detecting bases within a DNA background.
• Coverage: Sanger lower sample coverage compared to NGS.

Conclusion

• Sanger Sequencing: Remains a gold standard due to accuracy, despite the advent of NGS.
• Use Case: Ideal for small number of samples; NGS preferred for large-scale sequencing.