DNA Sequencing 1

Overview of DNA Replication
Chain Termination Sequencing
- Also known as:
- Sanger Sequencing
- Dideoxy Sequencing
- Cycle sequencing
Automated Sequencing
Readings
- Clarke Chapter 8
- Course Code: MMG2040 - 2026

Synthesis of DNA:
- The process involves four major phases: Priming, Elongation, Phosphodiester bond formation, and Termination.

Involves the linking of nucleotides wherein a phosphodiester bond is formed between the 5’ phosphate of one nucleotide and the 3’ hydroxyl of another.
- This continues to elongate the DNA strand.

Steps in Chain Termination Sequencing:
1. Isolate template DNA
- Methods include PCR, plasmid preparation, or genomic DNA preparation.
1. DNA replication conducted by DNA polymerase.
- Requires a primer to initiate.
1. Fluorescently labelled dideoxynucleotides (ddNTPs) are incorporated which halts the polymerization of the new DNA molecule.
2. Fragments are separated by size, and the fluorescent nucleotides are read by a sensor.
- Note: Previously, fragments were separated on a gel and detected by radiolabel.

Klenow Polymerase:
- The initial DNA polymerase used for sequencing.
- Originates from E. coli DNA Pol I.
- Klenow Fragment boasts the following features:
- Removed the 5’-3’ exonuclease activity.
- Exhibits low processivity (approximately 250 base pairs).
Sequenase:
- Originates from T7 phage.
- Higher processivity compared to Klenow polymerase.
Taq Polymerase:
- Originates from Thermus aquaticus.
- Essential for cycle sequencing.

Also known as the Klenow fragment.
Activities include:
- 5' → 3' Polymerase Activity: Facilitates addition of nucleotides to the growing DNA strand.
- 3' → 5' Exonuclease Activity: Enables proofreading ability, enhancing accuracy.
- Does NOT have a 5' → 3' exonuclease activity.

Functionality: ddNTPs are critical for terminating DNA strand elongation.
Unique Features: Each ddNTP is equipped with a distinctive fluorophore aiding in the detection process.
Fluorescent ddNTPs cannot be added to the growing chain. Their 3’-hydroxyl group is unavailable, which stops further elongation of the DNA strand.

Sequencing Primer:
- A short oligonucleotide complementary to the starting point of the target DNA.
- Provides a 3' hydroxyl group needed for DNA polymerase.
NTPs:
- A pool of deoxynucleoside triphosphates (dNTPs) and fluorescent ddNTPs is used.
- Normal dNTPs facilitate typical DNA elongation, while ddNTPs are incorporated to cause termination.
DNA Polymerase:
- Must be highly processive, allowing it to extend considerably before dissociating from the DNA template.
- Must also extend quickly and accurately to ensure fidelity in the sequencing process.

Primer Association with ddNTPs:
- 1) The primer is labeled with a radioisotope or fluorophore.
- 2) dNTPs are labeled and incorporated until the addition of ddNTPs instigates chain termination.
- 3) The termination caused by the incorporation of ddNTPs allows for sequencing of the complementary strand of the template DNA.

Diagrams and figures summarize the sequencing procedures and results (refer to Figures 8.01, 8.05, and 8.06).

Methodology:
- The fragments, once created through the chain termination process, are separated based on size via capillary electrophoresis.
- Visual representations demonstrate the movement of DNA fragments under the influence of an electric field.

Process Overview:
- Each sequencing reaction is conducted in a single capillary tube.
- Numerous capillary tubes (hundreds) can be utilized simultaneously to enhance throughput and efficiency.
- An electrical current propels DNA through the system.
- A laser excites the bound fluorophores.
- Emitted light is captured by a CCD camera.
- Computer systems interpret the fluorescence emissions and translate them into base identities.
- Capable of sequencing up to 700 bases per primer in a single run.