Sanger Sequencing

The Chemistry: dNTPs vs ddNTPs

dNTP (deoxynucleotide triphosphate):

• Normal DNA building blocks: dATP, dCTP, dGTP, dTTP
• Have a 3'-OH group → DNA polymerase can add another nucleotide
• Chain continues growing

ddNTP (dideoxynucleotide triphosphate):

• Modified nucleotides: ddATP, ddCTP, ddGTP, ddTTP
• Missing the 3'-OH group → no place to attach next nucleotide
• Chain terminates (stops growing)

The key idea: Mix normal dNTPs with a small amount of ddNTPs. Sometimes the polymerase adds a normal dNTP (chain continues), sometimes it adds a ddNTP (chain stops). This creates DNA fragments of different lengths, all ending at the same type of base.

The Classic Method: Four Separate Reactions

You set up four tubes, each with:

• Template DNA (what you want to sequence)
• Primer (starting point)
• DNA polymerase
• All four dNTPs (A, C, G, T)
• One type of ddNTP (different for each tube)

The Four Reactions:

Tube 1 - ddATP: Chains terminate at every A position Tube 2 - ddCTP: Chains terminate at every C position
Tube 3 - ddGTP: Chains terminate at every G position
Tube 4 - ddTTP: Chains terminate at every T position

Example Results:

Let's say the template sequence is: 5'-ACGTACGT-3'

Tube A (ddATP): Fragments ending at A positions

A
ACGTA
ACGTACGTA

Tube C (ddCTP): Fragments ending at C positions

AC
ACGTAC

Tube G (ddGTP): Fragments ending at G positions

ACG
ACGTACG

Tube T (ddTTP): Fragments ending at T positions

ACGT
ACGTACGT

Gel Electrophoresis Separation

Run all four samples on a gel. Smallest fragments move furthest, largest stay near the top.

        A    C    G    T
        |    |    |    |
Start → ━━━━━━━━━━━━━━━━  (loading wells)
        
        |              |  ← ACGT (8 bases)
        |    |         |  ← ACGTACG (7 bases)
        |              |  ← ACGTAC (6 bases) 
        |         |    |  ← ACGTA (5 bases)
        |         |    |  ← ACGT (4 bases)
        |    |    |    |  ← ACG (3 bases)
        |    |         |  ← AC (2 bases)
        |              |  ← A (1 base)
        
      ↓ Direction of migration ↓

Reading the sequence: Start from the bottom (smallest fragment) and go up:

Bottom → Top:  A - C - G - T - A - C - G - T
Sequence:      A   C   G   T   A   C   G   T

The sequence is ACGTACGT (read from bottom to top).

Modern Method: Fluorescent Dyes

Instead of four separate tubes, we now use one tube with four different fluorescent ddNTPs:

• ddATP = Green fluorescence
• ddCTP = Blue fluorescence
• ddGTP = Yellow fluorescence
• ddTTP = Red fluorescence

What happens:

1. All fragments are created in one tube
2. Run them through a capillary (tiny tube) instead of a gel
3. Laser detects fragments as they pass by
4. Computer records the color (= which base) and timing (= fragment size)

Chromatogram output:

Fluorescence
    ↑
    |     G  C     T     A  G  C  T
    |    /\  /\   /\    /\ /\ /\ /\
    |___/  \/  \_/  \__/  X  \/  \_____→ Time
    |                     / \
Position: 1  2  3  4  5  6  7  8

The computer reads the peaks and outputs: GCTAGCT

Why Sanger Sequencing Still Matters

• High accuracy (~99.9%)
• Gold standard for validating variants
• Good for short reads (up to ~800 bases)
• Single-molecule sequencing - no PCR bias
• Used for: Confirming mutations, plasmid verification, PCR product sequencing

Limitations:

• One fragment at a time (not high-throughput)
• Expensive for large-scale projects (replaced by next-gen sequencing)
• Can't detect low-frequency variants (< 15-20%)

About Course Materials

These notes contain NO copied course materials. Everything here is my personal understanding and recitation of concepts, synthesized from publicly available resources (textbooks, online tutorials, sequencing method documentation).

This is my academic work—how I've processed and reorganized information from legitimate sources. I take full responsibility for any errors in my understanding.

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