In DNA sequencing, which method is primarily used to determine the order of nucleotides?

Sanger sequencing is the method primarily used to determine the order of nucleotides in a DNA fragment. This technique, developed by Frederick Sanger, utilizes selective incorporation of chain-terminating dideoxynucleotides during DNA replication. By copying the DNA template in the presence of these modified nucleotides, fragments of varying lengths are generated. Each fragment ends at a specific nucleotide, allowing the sequence to be deduced by analyzing the lengths of the newly formed strands after separation, typically through capillary electrophoresis or gel electrophoresis.

In contrast, Western blotting is used to detect specific proteins in a sample, gel electrophoresis, while a technique that separates nucleic acids or proteins based on size, does not determine the sequence of nucleotides but rather is often used to analyze the results of sequencing. Southern blotting is also focused on DNA but is primarily used for the detection of specific DNA sequences within a complex mixture rather than sequencing them. Thus, Sanger sequencing stands out as the definitive method for determining nucleotide order in DNA.