Skip to main content
. 2016 Apr 4;113(16):E2277–E2285. doi: 10.1073/pnas.1600279113

Fig. S5.

Fig. S5.

Pyrophosphorolysis reactions with Vent pol. (A) Pyrophosphorolysis of DNA22 (A•T), DNA22 (C•T), and DNA22 (T•T). Of the four possible 22mer primers, pyrophosphorolysis is observed only on the correctly matched [DNA22 (A•T)] without the presence of dNTPs. Aliquots were quenched at 0 h, 12 h, 24 h, and 48 h. (B) Pyrophosphorolysis of DNA21 (G•C) in the presence of 32PPi. Pyrophosphorolysis reactions were carried out for 1 h or 40 h with carrier-free 32PPi to confirm dNTPs are the product of the pyrophosphorolysis reaction. After the reaction time, DNA and enzyme were removed using a 3-kDa cutoff filter. The filtrate was then incubated with yeast inorganic pyrophosphatase (YIPP) to turn any remaining PPi to inorganic phosphate (Pi), which is readily separated from dNTPs by TLC. After 1 h, a small spot corresponding to dNTPs is seen on the TLC plate, but by 40 h, these dNTPs have been reincorporated by Vent pol into the DNA and are no longer observed as a spot.