Sanger sequencing: The chain termination method
Regions
of DNA up to about 900900900 base pairs in length are
routinely sequenced using a method called Sanger sequencing or
the chain termination method. Sanger sequencing was
developed by the British biochemist Fred Sanger and his colleagues in 1977.
In
the Human Genome Project, Sanger sequencing was used to determine the sequences
of many relatively small fragments of human DNA. (These fragments weren't
necessarily 900900900 bp or less, but researchers were able to
"walk" along each fragment using multiple rounds of Sanger
sequencing.) The fragments were aligned based on overlapping portions to
assemble the sequences of larger regions of DNA and, eventually, entire
chromosomes.
Although
genomes are now typically sequenced using other methods that are faster and
less expensive, Sanger sequencing is still in wide use for the sequencing of
individual pieces of DNA, such as fragments used in DNA
cloning or
generated through polymerase chain reaction (PCR).
Ingredients for Sanger sequencing
Sanger sequencing involves
making many copies of a target DNA region. Its ingredients are similar to those
needed for DNA replication in an organism, or for polymerase
chain reaction (PCR), which copies DNA in vitro. They include:
- A DNA polymerase enzyme
- A primer,
which is a short piece of single-stranded DNA that binds to the template
DNA and acts as a "starter" for the polymerase
- The four DNA nucleotides (dATP,
dTTP, dCTP, dGTP)
- The template DNA to be sequenced
However, a Sanger sequencing
reaction also contains a unique ingredient:
- Dideoxy, or chain-terminating, versions of all four
nucleotides (ddATP, ddTTP, ddCTP, ddGTP), each labeled with a different
color of dye
Dideoxy nucleotides are similar
to regular, or deoxy, nucleotides, but with one key difference: they lack a
hydroxyl group on the 3’ carbon of the sugar ring. In a regular nucleotide, the
3’ hydroxyl group acts as a “hook," allowing a new nucleotide to be added
to an existing chain.
Once a dideoxy nucleotide has
been added to the chain, there is no hydroxyl available and no further
nucleotides can be added. The chain ends with the dideoxy nucleotide, which is
marked with a particular color of dye depending on the base (A, T, C or G) that
it carries.
Method of Sanger sequencing
The DNA sample to be sequenced
is combined in a tube with primer, DNA polymerase, and DNA nucleotides (dATP,
dTTP, dGTP, and dCTP). The four dye-labeled, chain-terminating dideoxy
nucleotides are added as well, but in much smaller amounts than the ordinary
nucleotides.
The mixture is first heated to
denature the template DNA (separate the strands), then cooled so that the primer
can bind to the single-stranded template. Once the primer has bound, the
temperature is raised again, allowing DNA polymerase to synthesize new DNA
starting from the primer. DNA polymerase will continue adding nucleotides to
the chain until it happens to add a dideoxy nucleotide instead of a normal one.
At that point, no further nucleotides can be added, so the strand will end with
the dideoxy nucleotide.
This process is repeated in a
number of cycles. By the time the cycling is complete, it’s virtually
guaranteed that a dideoxy nucleotide will have been incorporated at every
single position of the target DNA in at least one reaction. That is, the tube
will contain fragments of different lengths, ending at each of the nucleotide
positions in the original DNA (see figure below). The ends of the fragments
will be labeled with dyes that indicate their final nucleotide.
After the reaction is done, the
fragments are run through a long, thin tube containing a gel matrix in a
process called capillary gel electrophoresis. Short fragments
move quickly through the pores of the gel, while long fragments move more
slowly. As each fragment crosses the “finish line” at the end of the tube, it’s
illuminated by a laser, allowing the attached dye to be detected.
The smallest fragment (ending
just one nucleotide after the primer) crosses the finish line first, followed
by the next-smallest fragment (ending two nucleotides after the primer), and so
forth. Thus, from the colors of dyes registered one after another on the
detector, the sequence of the original piece of DNA can be built up one
nucleotide at a time. The data recorded by the detector consist of a series of
peaks in fluorescence intensity, as shown in the chromatogram above.
The DNA sequence is read from the peaks in the chromatogram.
Uses and limitations
Sanger sequencing gives
high-quality sequence for relatively long stretches of DNA .
It's typically used to sequence individual pieces of DNA, such as bacterial plasmids or DNA copied in PCR.
However, Sanger sequencing is expensive and inefficient for larger-scale projects, such as the sequencing of an entire genome or metagenome (the “collective genome” of a microbial community). For tasks such as these, new, large-scale sequencing techniques are faster and less expensive.
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