GENERAL KNOWLEDGE

A DEEP DIVE INTO THE SANGER METHOD FOR DNA SEQUENCING

The Sanger method, also known as the chain termination method, is a technique used to determine the sequence of nucleotides in a DNA molecule. This method was developed by Frederick Sanger and his colleagues in the late 1970s and has been fundamental in advancing our understanding of genetics, genomics, and molecular biology. The Sanger method revolutionized the field of DNA sequencing and laid the foundation for modern sequencing technologies.

Principles of the Sanger Method

The Sanger method relies on the synthesis of DNA in vitro using DNA polymerase and modified nucleotides. The key components of the Sanger sequencing reaction include a template DNA strand, a primer, DNA polymerase, deoxynucleotides (dNTPs), and dideoxynucleotides (ddNTPs). The ddNTPs lack a 3’ hydroxyl group necessary for the formation of a phosphodiester bond with the next nucleotide, leading to chain termination when they are incorporated into the growing DNA strand.

Procedure

  1. DNA Denaturation: The double-stranded DNA template is denatured into single strands by heating.
  2. Primer Annealing: A short synthetic oligonucleotide primer is annealed to the template DNA.
  3. DNA Synthesis: The primer is extended by DNA polymerase using both dNTPs and ddNTPs.
  4. Gel Electrophoresis: The synthesized fragments are separated by size using gel electrophoresis.
  5. Visualization: The separated fragments are visualized using autoradiography or fluorescent dyes.

Advantages and Limitations

The Sanger method has several advantages, including its accuracy, reliability, and ability to sequence relatively long stretches of DNA. However, it is labor-intensive, time-consuming, and limited in its throughput compared to high-throughput sequencing methods such as next-generation sequencing (NGS).

Impact and Legacy

The development of the Sanger method revolutionized genetics and molecular biology by enabling scientists to decipher the genetic code and sequence entire genomes. It played a pivotal role in numerous landmark scientific achievements, including the Human Genome Project.

Modern Applications

While newer sequencing technologies have largely supplanted the Sanger method for large-scale projects, it is still used in various applications such as targeted sequencing, validation of NGS data, and analyzing specific regions of interest in the genome.

In conclusion, the Sanger method for DNA sequencing has been instrumental in advancing our understanding of genetics and genomics. Its principles and techniques have paved the way for modern sequencing technologies and continue to be utilized in specific research applications.

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