EinzelgenSangerSequencing

EinzelgenSangerSequencing, also known as Sanger sequencing, is a method used to determine the precise order of nucleotides within a DNA molecule. Developed by Frederick Sanger and his colleagues in 1977, this technique is one of the earliest and most widely used methods for DNA sequencing. The process involves the use of DNA polymerase, which synthesizes a new strand of DNA complementary to the template strand, and a series of four differently colored fluorescently labeled dideoxynucleotides (ddNTPs). Each ddNTP is tagged with a different fluorescent dye, allowing for the identification of the nucleotide sequence.

The sequencing reaction is initiated by the addition of a primer to the template DNA, followed by the incorporation of ddNTPs by DNA polymerase. As the polymerase extends the new strand, it encounters a ddNTP, which lacks a 3'-hydroxyl group necessary for further elongation. This termination results in a population of DNA fragments of varying lengths, each ending with a different ddNTP. These fragments are then separated by size using capillary electrophoresis, and the fluorescent signals are detected as they pass through the detector. The sequence is determined by analyzing the order of the fluorescent peaks, which correspond to the nucleotides in the original DNA strand.

Sanger sequencing is highly accurate and has been instrumental in various fields, including genetics, genomics, and medical research. However, it has been largely superseded by newer, more efficient methods such as next-generation sequencing (NGS). Despite its decline in popularity, Sanger sequencing remains a valuable tool for specific applications where high accuracy and small-scale sequencing are required.