Developed by Frederick Sanger and his team in the late 1970s, Sanger sequencing revolutionized our ability to read DNA sequences accurately.
In the realm of DNA sequencing, this technique stands as one of the foundations that paved the way for modern genomic research, but how does Sanger sequencing work?
Join us as we embark on a journey through the inner workings of Sanger sequencing and uncover the key steps that make this technique an essential tool in molecular biology.
Primer Annealing: Setting the Stage
Sanger sequencing begins with a small piece of single-stranded DNA to be sequenced, which serves as the template.
This DNA fragment is mixed with a short synthetic oligonucleotide primer that anneals (binds) to a complementary sequence on the template. The primer acts as the starting point for DNA synthesis.
DNA Replication: Extending the Chain
Next comes the DNA replication phase. DNA polymerase, a naturally occurring enzyme, joins the party.
This enzyme extends the primer by adding complementary nucleotides to the template strand. These nucleotides are in the form of modified dideoxynucleotides (ddNTPs), which lack the 3′-OH group needed to form a phosphodiester bond with the next nucleotide, effectively halting further chain elongation.
Incorporating ddNTPs: A Clever Twist
Here’s the ingenious twist in Sanger sequencing: alongside regular deoxynucleotides (dNTPs) needed for DNA replication, a small fraction of ddNTPs (labeled with different fluorescent dyes) is included.
These ddNTPs, once incorporated, halt further chain elongation at specific positions.
Gel Electrophoresis: Separating the Strands
With the DNA fragments extended and labeled with fluorescent ddNTPs, it’s time for the separation.
The mixture is loaded onto a gel matrix, typically made of a thin slab of polyacrylamide. An electric current is applied across the gel, causing the DNA fragments to migrate based on size. Smaller fragments travel faster through the gel than larger ones.
Fluorescence Detection: Revealing the Sequence
As the fragments move through the gel, a laser excites the fluorescent labels on the ddNTPs.
Each ddNTP emits light at a characteristic wavelength, allowing a detector to capture the signal. The detector records the sequence as a series of peaks, with each peak representing a specific nucleotide in the DNA strand.
Data Analysis: Decoding the Sequence
The peaks obtained from the fluorescence detector are then processed through specialized software.
This software converts the peaks into a readable DNA sequence. The intensity and position of the peaks correspond to the identity and order of the nucleotides in the original DNA fragment.
In summary, Sanger sequencing is a cornerstone technique in molecular biology that allows scientists to read DNA sequences with high accuracy.
By combining primer annealing, DNA replication, ddNTP incorporation, gel electrophoresis, and fluorescence detection, researchers can decipher the genetic code. This method laid the foundation for the genomics revolution and remains a vital tool in various fields, from basic research to clinical diagnostics.