DNA Sequencing · Pathology

What is the primary purpose of using fluorescently labeled dideoxynucleotides (ddNTPs) in Sanger sequencing?

  1. To amplify the DNA fragment exponentially
  2. To specifically and permanently label the 5' end of the sequencing primer
  3. To terminate DNA synthesis at specific bases and label the fragments
  4. To provide energy for the DNA polymerase reaction
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Correct answer: To terminate DNA synthesis at specific bases and label the fragments

Dideoxynucleotides (ddNTPs) lack a 3'-OH group, which prevents the formation of a phosphodiester bond with the next incoming nucleotide, thereby terminating DNA strand elongation. When labeled with a fluorescent dye, they allow for the detection of the terminal base of each fragment via capillary electrophoresis.

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