3′- O -modified nucleotides as reversible terminators for pyrosequencing

Wu et al. 10.1073/pnas.0707495104.

Supporting Information

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SI Appendix
SI Figure 8
SI Figure 9
SI Figure 10
SI Table 1

SI Figure 8

Fig. 8. The polymerase extension scheme using 3'-O-(2-nitrobenzyl)-dTTP (Left) and MALDI-TOF MS spectra of the two consecutive extension products and their photocleavage products (Right). (A) Primer. (B) Primer extended with 3'-O-(2-nitrobenzyl)-dTTP to yield DNA extension product 2. (C) Product 2 photocleaved to yield photocleavage product 3. (D) Product 3 extended with another 3'-O-(2-nitrobenzyl)-dTTP to yield product 4. (E) Product 4 photocleaved to yield photocleavage product 5. After 30 sec of irradiation with a laser at 355 nm, photocleavage is completed with all of the 3'-O-(2-nitrobenzyl)-group cleaved from the extended DNA products.

SI Figure 9

Fig. 9. Experimental scheme for pyrosequencing with nucleotide reversible terminators [3'-O-allyl-dNTPs and 3'-O-(2-nitrobenzyl)-dNTPs; R = allyl or 2-nitrobenzyl]. A self-priming DNA template was immobilized on Sepharose beads. After the polymerase extension reaction is performed on the beads using 3'-O-modified nucleotides, the released PPi in the supernatant is detected with a luciferase enzyme kit to determine the identity of the incorporated nucleotide. The 3'-OH capping moiety from the DNA extension product is then removed to continue the subsequent polymerase reaction for the next sequence determination.

SI Figure 10

Fig. 10. Comparison of reversible terminator-pyrosequencing using 3'-O-(2-nitrobenzyl)-dNTPs with conventional pyrosequencing using natural nucleotides (NB = 2-nitrobenzyl). (A) Pyrosequencing using 3'-O-(2-nitrobenzyl)-dNTPs. In each cycle, the four reversible terminators are added iteratively and detected for light signal. After photocleavage, the next iterative addition cycle is performed. The addition of the complementary nucleotide yields the highest signal while the noncomplementary nucleotides produce much lower signals. (B) Pyrosequencing data using natural nucleotides. The exact sequence is difficult to decipher from the large peak corresponding to the homopolymeric adenines.

Table 1. Primer and template sequences for the incorporation of 3'-O-allyl-dNTPs and 3'-O-(2-nitrobenzyl)-dNTPs in solution

NRT	Primer sequence	Template sequence
3'-O-allyl-dATP	5'-TAGATGACCCTGCCTTGTCG-3'	Exon 8
3'-O-allyl-dCTP	5'-ACACAATAGAGGATCCAACCGAGA-3'	Exon 7
3'-O-allyl-dGTP	5'-GTTGATGTACACATTGTCAA-3'	Exon 7
3'-O-allyl-dTTP	5'-GTCAGCGCCGCGCCTTGGCGCGGCGC-3'*	SPT
3'-O-NB-dATP	5'-GACTGCGCCGCGCCTTGGCGCGGCGC-3'*	SPT
3'-O-NB-dCTP	5'-ATCGGCGCCGCGCCTTGGCGCGGCGC-3'*	SPT
3'-O-NB-dGTP	5'-GATCGCGCCGCGCCTTGGCGCGGCGC-3'*	SPT
3'-O-NB-dTTP	5'-GATAGGACTCATCACCA-3'	Exon 8

Exon 7 sequence: 5'-TACCCGGAGGCCAAGTACGGCGGGTACGTCCTTGACAATGTGTACATCAACATCACCTACCACCATGTCAGTCTCGGTTGGATCCTCTATTGTGTCCGGG-3'. Exon 8 sequence: 5'-GAAGGAGACACGCGGCCAGAGAGGGTCCTGTCCGTGTTTGTGCGTGGAGTTCGACAAGGCAGGGTCATCTAATGGTGATGATGCCTATCCTTTTCTCTTCGTTCTCCGT-3'. NB, 2-nitrobenzyl.

*These sequences are self-priming templates (SPT).