Sanger sequencing, also known as chain-termination sequencing, is a method used to determine the exact order of nucleotides in a DNA molecule. Developed by Frederick Sanger in 1977, it remains the gold standard for validating DNA sequences and detecting mutations.
How Sanger Sequencing Works
- Setting Up the Reaction
The DNA to be sequenced is mixed with a DNA primer, DNA polymerase, normal nucleotides (dNTPs), and a small amount of fluorescently labeled dideoxynucleotides (ddNTPs). Each of the four ddNTPs is labeled with a different fluorescent dye.
- Chain Termination
The reaction starts with the primer binding to the template DNA. The DNA polymerase extends the primer by adding dNTPs. Whenever a ddNTP is incorporated instead of a dNTP, the chain stops growing because ddNTPs lack the 3’-hydroxyl group needed for further extension.
- Fragment Generation
This process produces a mixture of DNA fragments of varying lengths, each ending at a specific nucleotide position. The fluorescent label on the terminating ddNTP identifies which base is at the end of each fragment.
- Capillary Electrophoresis
The mixture is loaded into a capillary electrophoresis instrument. The fragments are separated by size as they migrate through the capillary. A laser excites the fluorescent labels, and a detector records which color passes by at each time point.
- Chromatogram Analysis
The instrument produces a chromatogram—a series of colored peaks representing the sequence of nucleotides. The sequence is read from the chromatogram, typically from the shortest fragment to the longest, giving the complete DNA sequence.
