GENERAL KNOWLEDGE

OVERVIEW OF THE DIDEOXY METHOD OF SEQUENCING

The dideoxy method of DNA sequencing, also known as Sanger sequencing, is a widely used technique for determining the nucleotide sequence of DNA. This method was developed by Frederick Sanger and his colleagues in the late 1970s and has since become a fundamental tool in molecular biology and genetics.

Principle of Dideoxy Method of Sequencing

The principle behind the dideoxy method of sequencing lies in the use of chain-terminating dideoxynucleotides, also known as ddNTPs. These ddNTPs lack the 3’ hydroxyl group necessary for the formation of phosphodiester bonds with the next incoming nucleotide during DNA synthesis. When a ddNTP is incorporated into a growing DNA strand, it terminates further elongation, resulting in DNA fragments of varying lengths that collectively represent the entire sequence of the original DNA template.

Procedure

Here are the basic steps for the dideoxy method (Sanger sequencing):

  • DNA Denaturation: Heat the target DNA to separate the strands.
  • Primer Annealing: Cool the DNA to allow primers to anneal to the template strands.
  • DNA Synthesis: Use DNA polymerase, normal deoxynucleotides (dNTPs), and small amounts of dideoxynucleotides (ddNTPs) during DNA synthesis. The ddNTPs lack a 3′ hydroxyl group, leading to chain termination at their incorporation.
  • Fragment Separation: Separate the synthesized DNA fragments based on size using gel electrophoresis.
  • Detection: Visualize the separated fragments, often with a fluorescent dye or radioactive label.
  • Readout: Determine the sequence by identifying the terminated positions, which correspond to the ddNTPs in the DNA fragments.

Repeated cycles of these steps generate a series of fragments, revealing the sequence of the original DNA.

Advantages and Applications

The dideoxy method of sequencing offers several advantages, including its accuracy, reliability, and ability to sequence relatively long stretches of DNA. It has been instrumental in numerous scientific endeavors, including genome sequencing projects, identification of genetic mutations, forensic analysis, and evolutionary studies.

Challenges and Limitations

While highly effective, dideoxy sequencing does have limitations. It can be time-consuming and labor-intensive, especially when sequencing large genomes or multiple samples. Additionally, it may not be well-suited for high-throughput applications compared to more modern sequencing technologies such as next-generation sequencing methods.

In summary, the dideoxy method of sequencing has played a pivotal role in advancing our understanding of genetics and molecular biology. Its legacy continues to influence contemporary research and clinical practices in diverse fields.