Final answer:
The additions of dideoxynucleotide triphosphates (ddNTPs) by DNA polymerase during DNA sequencing are not random but are directed by the template DNA sequence. Each type of ddNTP is complementary to a DNA base and terminates chain elongation when incorporated. This process results in a collection of DNA fragments that can be sequenced to determine the template DNA sequence.
Step-by-step explanation:
The addition of nucleotides by DNA polymerase during the Sanger method of DNA sequencing involves the incorporation of deoxynucleotide triphosphates (dNTPs) and dideoxynucleotide triphosphates (ddNTPs). When a ddNTP is incorporated into the growing DNA strand, elongation is terminated because ddNTPs lack the necessary 3'-OH group required for forming the subsequent phosphodiester bond. This process is not random; rather, it is directed by the sequence of the template DNA that is being replicated. Each ddNTP is complementary to one of the DNA bases (A, T, G, C) and is added only when its complementary base is encountered in the template strand. Therefore, the incorporation of ddNTPs and the resulting DNA fragments reflect the original DNA sequence. Through this method, DNA polymerase adds nucleotides in a template-directed manner, and the selective incorporation of fluorescently labeled ddNTPs allows for the sequencing of the DNA.
During the sequencing process, a DNA sample is denatured and divided into four separate reactions, each containing a different fluorescently labeled ddNTP (ddCTP, ddATP, ddGTP, dnTTP), alongside the regular dNTPs and DNA polymerase. Chain elongation proceeds until a ddNTP is incorporated, at which point the chain is terminated. The resulting DNA fragments, each ending with a labeled ddNTP, are then separated by gel electrophoresis. Laser scanners detect the fluorescent labels to determine the sequence of the template DNA. This method, originally developed by Frederick Sanger, earned him a Nobel Prize in Chemistry in 1980.