Exploring the Sanger sequencing method: Its history and limitations

Sanger sequencing method

-Also referred to as dideoxy sequencing-Uses DNA polymerase-first generation sequencingRequires 3 steps:1) Cloning of the gene fragment to be sequenced2) DNA synthesis (using gene of interest as DNA template with help of DNA polymerase enzymes)3) Electrophoresis (separate the fragments of DNA)Sanger’s sequencing method is based on the fact that DNA polymerase requires a free 3′ hydroxyl group to continue DNA synthesis. The formation of phosphodiester bonds between two adjacent nucleotides requires a 3′ hydroxyl group. The hydroxyl group at the 3′ position is essential for chain elongation.The Deoxyribonucleoside triphosphate will become dideoxyribonucleoside triphosphate. (chain no longer grows)In the dideoxyribonucleoside, our Sanger method DNA synthesis process is terminated with a distinct, labeled end-point nucleotide. DNA sequence is then determined by detecting the labeled nucleotide at the end of each fragment, separated by gel electrophoresis.

The Sanger sequencing method (also known as dideoxy sequencing or chain termination sequencing) is a standard method used for DNA sequencing. It was developed by Frederick Sanger in the 1970s and revolutionized the field of genetics by drastically reducing the time and cost required for DNA sequencing.

The Sanger sequencing method is based on the use of DNA polymerase, a molecule that can make a copy of a single-stranded DNA template by adding matching nucleotides in a specific order. In traditional Sanger sequencing, four separate reactions are set up, each containing a single-stranded DNA template, a DNA polymerase, primers (short pieces of DNA that complement the template and provide a starting point for DNA synthesis), and fluorescently-labeled dideoxynucleotide triphosphates (ddNTPs).

In the presence of ddNTPs, DNA synthesis is halted because ddNTPs lack a 3′-OH group, which is required for the formation of the next phosphodiester bond. This results in the production of a series of DNA fragments of varying lengths, each ending in a fluorescently-labeled ddNTP.

The DNA fragments are then run through a process called gel electrophoresis, which separates them based on their length. Finally, the resulting gel image is analyzed using specialized software to determine the sequence of nucleotides.

The Sanger sequencing method has been widely used for a variety of applications, including genome sequencing, identification of genetic mutations, and evolutionary studies. However, the method has certain limitations, such as the inability to sequence long stretches of DNA and a high error rate in certain regions of the genome. These limitations have led to the development of newer, more advanced sequencing technologies, such as next-generation sequencing.

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