In-Depth Notes on DNA Sequencing and Sanger Sequencing Techniques

DNA Sequencing Overview

• The study of determining the sequence of nucleotides in DNA.

Human Genome Project

• Genome Definition: Complete set of DNA in an organism.
• Human Genome Size: Approximately 3 billion base pairs.
• Objective: International project (1998-2003) aimed to sequence and map all human genes (Homo sapiens).

Sanger Sequencing

• Method Used: Dideoxy termination sequencing method (also known as chain termination method).
• **Components: **
  • dNTPs (deoxynucleoside triphosphates) are the building blocks for DNA.
  • Replaced by ddNTPs (dideoxyribonucleotides) which terminate DNA synthesis when incorporated.
• Challenge: When ddNTPs are used, they stop the replication process upon incorporation because they lack the 3' hydroxyl (' OH) group necessary for further addition of nucleotides.

Dideoxyribonucleotides (ddNTPs)

• Function: When randomly incorporated into a DNA strand, they halt synthesis immediately.
• Illustration: A primer with a 5' phosphate group can be extended with normal dNTPs until a ddNTP is added.

Sanger Sequencing Process

• Requirements:
  • Template: Single-stranded DNA.
  • Primer: Radioactively labeled primer attached to the DNA template.
  • Polymerase: DNA polymerase to aid in nucleic acid synthesis.
  • Nucleotide Triphosphates: Regular dATP, dTTP, dGTP, and dCTP are used for normal DNA elongation.

Dideoxy Method Steps

1. Setup: Primed DNA added to four separate reaction tubes with DNA polymerase and dNTPs.
2. Incorporation of ddNTPs: Each tube contains a different labeled ddNTP in low concentration (A, T, C, G).
3. Chain Termination: ddNTPs prevent further polymerization, resulting in DNA fragments of varying lengths.
4. Electrophoresis: DNA fragments separated by size using polyacrylamide gel electrophoresis, allowing sequence reading based on length.

Sanger Sequencing Setup

• Controlled proportions in each tube:
  • Tube A-D: Each tube has dATP, dGTP, dTTP, dCTP, and one ddNTP.
  • Tube A: 5% ddATP
  • Tube B: 5% ddGTP
  • Tube C: 5% ddTTP
  • Tube D: 5% ddCTP

Example of Fragment Analysis

• Sequence reading processes involve identifying the last incorporated nucleotide for each fragment, determining the sequence order from the gel.

Automated Sequencing

• Advancement: Large-scale sequencing improvements.
• Detection: Each reaction is linked to a different fluorescent dye, allowing simultaneous readouts.
• Results: Combined samples run through a single gel lane, read by computer systems that output an electropherogram displaying fluorescent signals from each base.

Modern Sequencing Techniques

• Example Sequence: Modern sequencing outputs condense millions of base pairs into manageable readout formats, allowing for high-throughput analysis of genomic data.
• Efficient for comprehensive genomic studies and applications in research and medicine.

Conclusion

• Mastering these techniques, including PCR, RFLP, and dideoxy sequencing, is crucial for advancement in genetic research and biotechnological applications.