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. 2005 Jun 2;33(10):3176–3184. doi: 10.1093/nar/gki628

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© The Author 2005. Published by Oxford University Press. All rights reserved

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Pyrosequencing of ODNs containing d^MeisoC and disoG. All primer binding regions were identical and a 9 nt template region was varied. Nucleotide dispensations are indicated on the x-axis and relative peak height of light emitted from pyrophosphate release is the y-axis. Consecutive dispensations of a single nucleotide are cumulatively tallied in a single bar. At least one negative control dispensation of a non-complementary nucleotide was made during pyrosequencing of each ODN. These negative control dispensations resulted in no pyrophosphate release and have been omitted for clarity. (A) A template sequence of all natural nucleobases, 3′-CTATGTATC-5′, was sequenced as a positive control. (B) Template sequences containing d^MeisoC, such as 3′-CATAiCATAC-5′, displayed complete incorporation of disoG, but extension at subsequent natural nucleobases was inhibited. (C) Allowing more time for incorporation at template positions following d^MeisoC through consecutive dispensations of a single nucleotide improved the incomplete incorporation observed in (B). (D) Template sequences containing disoG, such as 3′-CTGTiGTGTC-5′, displayed incomplete incorporation of d^MeisoC nucleotide, and extension at subsequent natural nucleobases was inhibited. (E) Consecutive dispensations of d^MeisoCTP opposite the template disoG position in (D) incorporated slightly more nucleotide, but still gave less than one equivalent of nucleotide incorporation. Consecutive dispensations of the same nucleotide at positions following disoG improved the incomplete incorporation at these positions observed in (D). (F) Template sequence 3′-CTGTiCTGTC-5′ demonstrated misincorporation of disoG nucleotide opposite template dT following correct incorporation opposite template d^MeisoC. (G) Dispensing a 1:1 mixture of disoG and dA nucleotides in place of disoGTP in (F) generated no pyrophosphate at the following dispensation of dA nucleotide and improved incorporation at the second position following the template d^MeisoC position, suggesting correct incorporation opposite template d^MeisoC and the following dA position.