Sanger sequencing and dideoxynucleotide chain termination
Sanger sequencing reads the exact order of nucleotides in DNA, using special building blocks called dideoxynucleotides that stop the new chain at a known base.
Remember
Once a dideoxynucleotide is built in, the chain stops growing, because it has no 3′ hydroxyl group (the OH on the sugar where the next nucleotide would attach). Each fragment stops at a known base, and reading the fragments in order of size gives the sequence.
Memory aid
no 3′ OH means no handle for the next nucleotide, so the chain is dead-ended
The missing OH is a missing handle: with nothing to hold, the chain is dead-ended.
DNA polymerase (the enzyme that copies DNA) builds a new chain from a primer (a short starting strand). To attach the next nucleotide, it needs a free 3′ hydroxyl group, the OH on the sugar at the end of the chain.
In Sanger sequencing, the reaction holds mostly ordinary nucleotides plus a small proportion of dideoxynucleotides. These lack the 3′ hydroxyl group. When the polymerase happens to add one, the chain offers no point of attachment, so copying stops at that base.
A dideoxynucleotide goes in at random positions, so the copies stop at different lengths. The mixture holds fragments that differ in length by one nucleotide, each stopping at a known base. Each of the four bases has its own fluorescent label (a dye) on its dideoxynucleotide, so the last base of every fragment can be identified by its colour.
The fragments are sorted by size, from shortest to longest, and the labels are read in that order. This spells out the sequence one base at a time.
That base-by-base read-out is why Sanger sequencing suits a single-base substitution, a change in one nucleotide of the DNA sequence. The sequence from the patient is compared with the normal sequence and the changed letter shows, even if only one of the two copies of the gene (one allele) carries it. The region is usually amplified first by PCR (polymerase chain reaction, which makes many copies of a chosen stretch of DNA) so that there is enough DNA to read.
Key facts
- Normal extension
- DNA polymerase adds the next nucleotide to a free 3′ hydroxyl group on the chain.
- The terminator
- a dideoxynucleotide has no 3′ hydroxyl group, so no further nucleotide can be added once it is in the chain.
- The mixture
- mostly ordinary nucleotides, with a small proportion of dideoxynucleotides.
- The fragments
- each one ends in a known base, and they differ in length by one nucleotide.
- The read-out
- fragments are sorted by size, from shortest to longest, and their labels are read in order to give the sequence.
- Well suited to
- confirming a single-base substitution in a defined region of DNA, even when only one allele carries it.
- Usual workflow
- PCR amplifies the region first, and sequencing then reads it.
Exam traps
- A 2′ hydroxyl group does not stop the chain. A ribonucleotide (the building block of RNA) carries one but keeps its 3′ hydroxyl group as well, so the missing 3′ hydroxyl group of the dideoxynucleotide is what ends the chain.
- The fluorescent dye does not block the active site of DNA polymerase. The dye lets the last base of each fragment be identified by its colour, while termination comes from the missing 3′ hydroxyl group.
- Proofreading exonuclease activity, which removes a wrongly paired nucleotide from the end of an existing strand, is not how a dideoxynucleotide works. The chain stops because it cannot be extended, not because the added nucleotide is cut out.
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