A Sanger is a type of DNA sequencing method named after its inventor, Frederick Sanger. It is a widely used technique for determining the order of nucleotides in a DNA molecule.
How does Sanger Sequencing work?
Sanger sequencing relies on the principle of chain termination. The process involves:
- DNA template preparation: The DNA to be sequenced is first copied multiple times using a process called PCR.
- Primer annealing: A short piece of DNA called a primer binds to a specific location on the template DNA.
- DNA polymerase activity: DNA polymerase uses the primer as a starting point to extend a new DNA strand.
- Chain termination: The key to Sanger sequencing is the use of dideoxynucleotides (ddNTPs). These are modified nucleotides that lack a 3'-hydroxyl group, preventing further DNA extension. Each ddNTP is labeled with a different fluorescent dye.
- Separation and detection: The newly synthesized DNA fragments are separated by size using capillary electrophoresis. As the fragments pass through a laser beam, the fluorescent dyes are excited, and the emitted light is detected by a camera. This allows the sequence of nucleotides to be determined based on the order of the fluorescent signals.
Advantages of Sanger Sequencing:
- High accuracy: Sanger sequencing is known for its high accuracy, typically exceeding 99%.
- Versatility: It can be used to sequence both short and long DNA fragments.
- Well-established: Sanger sequencing is a well-established and reliable technique with a long history of use.
Applications of Sanger Sequencing:
- Genetic diagnostics: Sanger sequencing is commonly used for identifying mutations associated with genetic diseases.
- Forensic science: It helps in identifying individuals from DNA samples.
- Basic research: Sanger sequencing is used for studying gene function and evolution.
Practical Insights:
- Sanger sequencing is commonly used in laboratories for research and diagnostic purposes.
- The cost of Sanger sequencing has decreased significantly in recent years, making it more accessible.
- While newer sequencing technologies are available, Sanger sequencing remains a valuable tool for specific applications.
