The Sanger sequencing method is a technique that allows the determination of the sequence of DNA nucleotides within a strand of DNA. This method is based on the incorporation of dideoxynucleotides, which are modified nucleotides that lack the 3' hydroxyl group necessary for the formation of phosphodiester bonds with the next nucleotide.
The process of Sanger sequencing begins with the preparation of a DNA template strand that is to be sequenced. This template strand is then mixed with a DNA polymerase enzyme, four types of deoxynucleotides (dATP, dCTP, dGTP, and dTTP), and a small amount of one of the four dideoxynucleotides (ddATP, ddCTP, ddGTP, or ddTTP). The dideoxynucleotide is labeled with a fluorescent dye to distinguish it from the deoxynucleotides.
The polymerase enzyme will incorporate the deoxynucleotides into a newly synthesized strand of DNA until it incorporates the modified dideoxynucleotide, which lacks the 3' hydroxyl group required for forming the next phosphodiester bond. Due to this deficiency, the incorporation of the dideoxynucleotide terminates strand elongation.
This reaction generates fragments of varying lengths, each ending with a dideoxynucleotide at its 3' end. These fragments are then separated by size using a gel electrophoresis apparatus based on their changes in electrophoretic mobility. The resulting bands of fragments are then analyzed using a laser scanner or fluorescence detector that identifies the color of the attached dye.
This technique produces a series of fluorescent peaks corresponding to the sequence of nucleotides within the original DNA strand. The order of fluorescent peaks indicates the sequence of the DNA strand, with each peak representing a specific nucleotide in the sequence, and the length of the fragments representing the location of each nucleotide within the original strand.
Describe how a dna strand is sequenced with the sanger sequencing method
1 answer