Sangersekvensointia

Sangersekvensointia, also known as chain-termination sequencing, is a method for determining the nucleotide sequence of DNA. Developed by Frederick Sanger in 1977, it was the first widely used DNA sequencing method and remained the gold standard for decades. The technique relies on the selective incorporation of chain-terminating dideoxynucleotides during DNA replication.

The process begins with a DNA template, a primer, DNA polymerase, and a mixture of all four deoxyribonucleotides (dNTPs). In addition to the regular dNTPs, the reaction includes a small amount of modified nucleotides called dideoxynucleotides (ddNTPs). Each ddNTP is labeled with a unique fluorescent dye. When a ddNTP is incorporated into the growing DNA strand, it terminates elongation because it lacks the 3'-hydroxyl group necessary for phosphodiester bond formation.

The reaction is typically performed in four separate tubes, each containing a different ddNTP (ddATP, ddTTP, ddCTP, or ddGTP), along with all four dNTPs. Alternatively, a single tube can be used with each ddNTP labeled with a different colored fluorescent dye. After the polymerase extends DNA strands from the primer, a collection of DNA fragments of varying lengths is generated, each ending with a specific ddNTP.

These fragments are then separated by size using gel electrophoresis or capillary electrophoresis. In the traditional method, this separation results in distinct bands on a gel that can be read manually. With fluorescently labeled ddNTPs, a detector reads the fluorescent signal as each fragment passes by, producing a chromatogram that represents the DNA sequence. Sanger sequencing is highly accurate for individual DNA fragments and is still used for validation of sequencing results from other technologies.