What is NOT a requirement for Sanger Sequencing?

In Sanger Sequencing, a key characteristic is the requirement for a DNA template that must be single-stranded. This allows DNA polymerase to synthesize a complementary strand by adding nucleotides to the growing DNA chain. To facilitate this synthesis, both normal deoxynucleosidetriphosphates (dNTPs) and a suitable DNA polymerase are essential.

However, the use of an RNA primer does not apply to Sanger Sequencing. Instead, a DNA primer is used to initiate the synthesis of the new DNA strand. This is crucial as the DNA polymerase requires a primer with a free 3'-OH group to begin adding nucleotides. Thus, while the other components (single-stranded DNA template, DNA polymerase, and dNTPs) are necessary for the sequencing process, the use of an RNA primer is not a requirement and is what makes this answer correct.