Sangersekvensoinnin

Sangersekvensoinnin, also known as the chain-termination method or dideoxy method, is a foundational DNA sequencing technique developed by Frederick Sanger in 1977. It was the first widely used and highly efficient method for determining the precise order of nucleotides within a DNA molecule. The core principle of Sanger sequencing relies on the selective incorporation of modified nucleotides called dideoxynucleotides (ddNTPs) during DNA synthesis.

In the Sanger method, a DNA sample is denatured into single strands and then subjected to a DNA polymerase reaction. This reaction includes a DNA primer, a DNA polymerase enzyme, normal deoxynucleotides (dNTPs), and a small amount of fluorescently labeled dideoxynucleotides (ddATP, ddTTP, ddCTP, ddGTP). When a ddNTP is incorporated into the growing DNA strand, it prematurely terminates the synthesis because it lacks the 3'-hydroxyl group necessary for forming phosphodiester bonds.

This process results in a collection of DNA fragments of varying lengths, each ending with a specific ddNTP. These fragments are then separated by size, typically using gel electrophoresis or capillary electrophoresis. As the fragments migrate, a detector reads the fluorescent signal emitted by the ddNTP at the end of each fragment. The order of these signals corresponds to the nucleotide sequence of the original DNA.

Although newer sequencing technologies like next-generation sequencing have surpassed Sanger sequencing in throughput and cost-effectiveness for large-scale projects, Sanger sequencing remains valuable for specific applications. These include sequencing individual genes, verifying results from other methods, and sequencing short DNA fragments. Its reliability and accuracy have made it a cornerstone of molecular biology research and diagnostics for decades.