Clarifying the Sanger Method of DNA Sequencing: Principles and Key Components

Understanding the Sanger Method of DNA Sequencing

The Sanger method sequences DNA by incorporating both normal nucleotides (dNTPs) and special chain-terminating nucleotides (ddNTPs) into a DNA synthesis reaction, creating fragments of varying lengths that end with fluorescently labeled ddNTPs. These fragments are separated by size allowing determination of the original DNA sequence based on the order and color of fragment termination.

Principle of Chain Termination

The Sanger sequencing reaction mixes abundant normal deoxynucleotides (dNTPs) with a smaller amount of dideoxy nucleotides (ddNTPs). DNA polymerase extends a primer along the template strand by adding dNTPs.

When a ddNTP incorporates, it lacks a 3′-OH group required to form a phosphodiester bond, terminating further extension. Each ddNTP is tagged with a different fluorescent dye, marking the base where synthesis stops.

• This stochastic incorporation creates a series of DNA fragments ending at each position where a ddNTP was inserted.
• Fragments reflect all possible termination points along the template.

Separation and Sequential Reading

Fragments are separated by length using electrophoresis. Because the shortest fragments migrate fastest, the sequence can be read from the order in which colored fragments elute.

• The shortest fragments emerge first, corresponding to positions closest to the primer.
• Fluorescent signals are detected in order, revealing the DNA sequence one base at a time.
• Early bases (first 50–100) may have unreliable readings due to erratic fragment behavior.

Size separation must be precise. Without accurate resolution by length, fragment data becomes unreadable, preventing sequence identification.

Interpreting Fragment Data

Fragments are noted by their terminating ddNTP (uppercase letter) and preceding nucleotides (lowercase). For example, from a sequence 3′-aattccga-5′, fragments like 3ttaaG5 indicate termination at G with preceding nucleotides seen.

Even if a long fragment forms first, the presence of all other shorter fragments helps reconstruct the entire sequence by assembling all fragments logically in order.

Essential Components and Directionality

Sanger sequencing requires a primer complementary to a known DNA region just before the target sequence.

• Extension occurs in one direction only, adding nucleotides sequentially.
• Each added nucleotide increases fragment length by one, allowing position mapping relative to the primer.
• Position 1 corresponds to the first base added past the primer; position 1000 is the primer length plus 1000 nucleotides.

Key Takeaways

• Sanger sequencing relies on chain-terminating ddNTPs labeled with distinct fluorescent dyes.
• Fragment sizes vary due to random ddNTP incorporation; size separation reveals sequence order.
• Accurate size separation is crucial for reliable sequence determination.
• Primers define the start point and direction for extension and sequencing.
• Fragment data assembled by termination base and length reconstructs the original DNA sequence.