Question

You can improve the read length of Sanger Sequencing by changing the enzymatic activity of the original DNA Polymerase.

a. Explain the problems with the DNA polymerase and the modifications required. [3 M]

b. Name a commercial Sanger Sequencing DNA polymerase with properties in a) [1 M]

Answer 1

A. Polymerase I effectively discriminates between a deoxy- and dideoxyribose in the nucleoside triphosphate and does not incorporate ddNTPs.
2. The incorporation rate of ddNTP by Pol I is several hundred-fold slower than that of normal dNTPs.
3. The sequence-specific ddNTP incorporation by Pol I creates non-uniform band intensities on the gel.

B. T4 DNA polymerase.

It incorporates both types of nucleotides at nearly equal efficiencies.
The intensities of dideoxy-terminated bands are significantly more uniform.