Sanger sequencing, a method used to determine the nucleotide sequence of a DNA molecule, uses a mix of dNTPs and a chain terminating di-deoxy NTP in a PCR reaction. The structure below shows ddGTP. Which of the following arrows correctly represents the site where DNA polymerase would typically add the next nucleotide?
- I
- II
- III
- IV
Click for Explanation
B is correct. DNA polymerase builds nucleotide chains in the 5’ to 3’ direction, meaning that the next nucleotide is added to the 3’ OH group of the deoxyribose sugar. In the molecule above, II refers to the 3’ carbon and III refers to the 2’ sugar. DNA nucleotides will normally have a 3’ OH but lack a 2’ OH (the difference between deoxyribose from ribose). The molecule above has a di-deoxy ribose sugar and lacks both OH groups. Without a 3’ OH group, DNA polymerase cannot add the next nucleotide, which terminates the extension of the strand.
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