Dideoksimenetelmäksi

Dideoksimenetelmäksi refers to a method for determining the nucleotide sequence of a DNA fragment. This technique, also known as the Sanger sequencing method or chain-termination sequencing, was developed by Frederick Sanger and his colleagues. It relies on the use of dideoxynucleotides, which are modified versions of the normal deoxynucleotides that make up DNA.

In the dideoksimenetelmäksi process, DNA replication is initiated using a DNA polymerase enzyme, a primer, and a mixture of normal deoxynucleotides (dNTPs). The key to the method is the inclusion of small amounts of dideoxynucleotides (ddNTPs). When a dideoxynucleotide is incorporated into a growing DNA strand, it terminates the elongation because it lacks the 3'-hydroxyl group necessary for forming the next phosphodiester bond.

The reaction is typically carried out in four separate tubes, each containing the DNA template, primer, DNA polymerase, and all four dNTPs, but with a different dideoxynucleotide (ddATP, ddTTP, ddCTP, or ddGTP) added to each. This results in a collection of DNA fragments of varying lengths, each ending with a specific dideoxynucleotide. These fragments are then separated by size, usually using gel electrophoresis. The order of the fragments on the gel reveals the sequence of the DNA. While originally labor-intensive, automated versions using fluorescently labeled dideoxynucleotides have made dideoksimenetelmäksi a widely used and efficient method for DNA sequencing.