Practice question

Sanger sequencing employs dideoxynucleoside triphosphates, ddNTPs, as essential terminators alongside normal dNTPs. The reaction contains all four dNTPs for strand extension plus limiting concentrations of labeled ddATP, ddTTP, ddGTP and ddCTP in four separate or single combined reactions. DNA polymerase synthesizes complementary strand until a ddNTP is randomly incorporated. Since ddNTPs lack a 3' hydroxyl group, further phosphodiester bond formation is impossible and synthesis halts permanently. Ribonucleotides rNTPs are not used, and dATP alone is insufficient for comprehensive sequence determination.