The Sanger dideoxynucleotide method, also known as Sanger sequencing or the chain-
termination method, is a foundational technique for determining the order of nucleotide bases (A,
T, C, G) in a DNA molecule. Developed by Frederick Sanger in 1977, it was the gold standard for
DNA sequencing for decades and played a crucial role in the Human Genome Project.
The core principle of Sanger sequencing relies on the use of dideoxynucleotides (ddNTPs). These
are modi ed nucleotides that lack a hydroxyl (-OH) group at the 3' carbon of their sugar ring. In
normal DNA synthesis, the 3'-OH group is essential for forming a phosphodiester bond with the
incoming nucleotide, allowing the DNA strand to elongate. When a ddNTP is incorporated into a
growing DNA strand, elongation is immediately terminated because there's no 3'-OH group to form
the next phosphodiester bond.
Here's a breakdown of the method:
1. Materials:
• DNA template: The DNA fragment whose sequence you want to determine (single-
stranded).
• Primer: A short, single-stranded DNA sequence that is complementary to a known region of
the template DNA. This primer serves as a starting point for DNA synthesis.
• DNA polymerase: An enzyme that synthesises new DNA strands by adding nucleotides.
• Deoxynucleotides (dNTPs): The four standard building blocks of DNA (dATP, dGTP,
dCTP, dTTP). These are present in excess.
• Dideoxynucleotides (ddNTPs): Modi ed nucleotides (ddATP, ddGTP, ddCTP, ddTTP).
These are present in much smaller amounts than dNTPs and are typically labelled (either
radioactively or, more commonly, uorescently).
2. The Reaction (Traditional Method - Four Separate Reactions): Historically, Sanger
sequencing involved setting up four separate reaction tubes, each containing:
• The DNA template
• The primer
• DNA polymerase
• All four dNTPs
• One type of ddNTP (e.g., tube 1 has ddATP, tube 2 has ddGTP, tube 3 has ddCTP, tube 4
has ddTTP).
In each tube, DNA polymerase synthesises new DNA strands from the primer, using the dNTPs.
However, because a small amount of ddNTPs is also present, there's a chance that a ddNTP will be
incorporated instead of a dNTP. When this happens, DNA synthesis stops at that point. This leads to
a collection of DNA fragments of varying lengths, all ending with the speci c ddNTP added to that
tube.
3. Fragment Separation and Detection (Traditional Method):
• After the reactions are complete, the DNA fragments from each of the four tubes are
separated by size using gel electrophoresis (typically polyacrylamide gel electrophoresis).
The fragments from each tube are loaded into separate lanes on the gel.
• Smaller fragments travel faster through the gel than larger fragments.
• After electrophoresis, the gel is analyzed (e.g., by autoradiography if radioactive labels were
used, or by UV light if uorescent labels were used).
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• By reading the bands from bottom to top (smallest to largest fragment) across the four lanes,
the sequence of the DNA can be deduced. For example, if a band appears in the "A" lane at
a certain position, it means an "A" was at that position in the original DNA sequence.
4. Automated Sanger Sequencing (Modern Method - Dye-Terminator Sequencing): Modern
Sanger sequencing is highly automated and uses dye-terminator sequencing. This method is more
ef cient and involves:
• Performing a single reaction with all four ddNTPs included in the same tube.
• Each of the four ddNTPs is labeled with a unique uorescent dye that emits light at a
different wavelength (e.g., ddATP = green, ddGTP = yellow, ddCTP = blue, ddTTP = red).
• The resulting fragments, each ending with a speci c dye-labeled ddNTP, are then separated
by size using capillary electrophoresis. As the fragments pass a detector, a laser excites the
uorescent dyes, and the emitted light is detected.
• A computer then records the color sequence, which directly translates into the DNA
sequence. This data is displayed as a chromatogram, a graph showing peaks of different
colors, each peak representing a nucleotide.
Applications:
Despite the advent of Next-Generation Sequencing (NGS), Sanger sequencing remains valuable for:
• Validating NGS results: It's often considered the "gold standard" for accuracy in certain
applications.
• Sequencing single genes or targeted regions: It's cost-effective for smaller sequencing
projects.
• Diagnostic applications: Identifying speci c mutations or genetic variations associated
with diseases.
• Con rmation of cloned inserts or site-directed mutagenesis: Verifying the success of
molecular cloning or genetic engineering experiments.
• Forensics and paternity testing: Its high accuracy makes it suitable for these applications.
Advantages:
• High accuracy (especially for single-nucleotide variants and small insertions/deletions).
• Can produce relatively long reads (up to 800-1000 base pairs).
• Less computationally intensive than NGS for analysis.
Limitations:
• Lower throughput compared to NGS (cannot sequence many genes or samples in parallel
ef ciently).
• More expensive for large-scale sequencing projects (whole genomes, exomes).
• May not detect mosaicism (presence of different cell lines with different genetic sequences
within an individual).
• Requires a relatively pure and high-quality DNA sample.
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