Sanger mail

You're referring to Sanger sequencing, a method used in molecular biology to determine the order of nucleotides (A, C, G, and T) in a DNA sequence.

Sanger sequencing is a dideoxy chain termination method developed by Frederick Sanger and his colleagues in the 1970s. It's a widely used technique for sequencing DNA fragments, including genes, genomes, and plasmids.

Here's a brief overview of the process:

  1. Template preparation: A DNA fragment is prepared as a template for sequencing. This can be done by PCR (polymerase chain reaction) amplification or by isolating the DNA fragment from a biological sample.
  2. Primer annealing: A primer is designed to bind specifically to the template DNA at a known location. The primer is annealed to the template DNA by heating and cooling the mixture.
  3. Dideoxynucleotide incorporation: A mixture of dideoxynucleotides (ddNTPs) is added to the reaction mixture. These nucleotides are similar to regular nucleotides but lack a 3'-OH group, which prevents further chain elongation.
  4. DNA polymerase extension: DNA polymerase is added to the reaction mixture, and it extends the primer by incorporating nucleotides into the growing DNA chain. The ddNTPs are incorporated randomly, terminating the chain at specific points.
  5. Separation by size: The reaction mixture is then separated by size using polyacrylamide gel electrophoresis (PAGE). The DNA fragments are separated based on their length, with the shortest fragments migrating the farthest.
  6. Detection: The separated DNA fragments are then detected using autoradiography or fluorescence. The presence of a band at a specific size indicates the presence of a particular nucleotide sequence.
  7. Sequence assembly: The sequence is assembled by analyzing the size and position of the bands on the gel. The sequence is read from the top of the gel to the bottom, with each band representing a single nucleotide.

Sanger sequencing has been widely used for many years and has played a crucial role in many scientific discoveries, including the Human Genome Project. However, with the advent of next-generation sequencing (NGS) technologies, Sanger sequencing has largely been replaced by more efficient and cost-effective methods.