A Sequencing Method Based on Real-Time Pyrophosphate

10.1126/science.7542800

Fraser C. M., et al., ibid. 270, 397 (1995);

Bult C. J., et al., ibid. 273, 1058 (1996);

10.1038/41483

Mewes H. W., et al., ibid. 387, 7 (1997).

10.1126/science.270.5235.484

10.1073/pnas.74.12.5463

Drmanac R., Labat I., Brukner I., Crkvenjakov R., Genomics4, 114 (1989);

Köster H., et al., Nature Biotechnol. 14, 1123 (1996).

Hyman E. D., Anal. Biochem.174, 423 (1988).

Rosenthal A. International Patent Application Publication 761107(1989).

Metzker M. L. , et al., Nucleic Acids Res.22, 4259 (1994).

Jones D. H., Biotechniques22, 938 (1997).

Ronaghi M., Karamohamed S., Pettersson B., Uhlén M., Nyrén P., Anal. Biochem.242, 84 (1996).

Nyrén P., Lundin A., ibid.151, 504 (1985).

The oligonucleotides E3PN (5 -GCTGGAATTCGTCAGACTGGCCGTCGTTTTACAAC-3) NUSPT (5 -GTAAAACGACGGCCAGT-3) and JA80 (5 -GATGGAAACCAAAAATGATAGG-3) were synthesized by phosphoramidite chemistry (Interactiva).

The oligonucleotide E3PN and the PCR product generated from cloned HIV-V3 were used as templates for DNA sequencing. The oligonucleotides and single-stranded PCR product were hybridized to the primers NUSPT and JA80 respectively. The hybridized DNA fragments were incubated with exo- Klenow or exo- T7 DNA polymerase (Sequenase 2.0) respectively (Amersham). The sequencing procedure was carried out by stepwise elongation of the primer-strand upon sequential addition of the different deoxynucleoside triphosphates and simultaneous degradation of nucleotides by apyrase (nucleoside 5 -triphosphatase and nucleoside 5 -diphosphatase;EC 3.6.1.5) (Sigma). The sequencing reaction was performed at room temperature and was started by adding a specific amount of one of the deoxynucleotides. The PPi released due to nucleotide incorporation was detected as described (9).

Ronaghi M. Nyrén P. data not shown.

The biotinylated PCR products were immobilized onto streptavidin-coated super paramagnetic beads [Dynabeads M280-Streptavidin (Dynal)]. Elution of single-stranded DNA and hybridization of sequencing primers was carried out as described (9).

The sequencing data obtained from the pyrosequencing method was confirmed by semiautomated solid-phase Sanger sequencing (18).

Doremus H. D., Belvins D. G., Plant Physiol87, 36 (1988).

The pyrosequencing instrument was based on a cassette containing the four separate nucleotides on an x-ray robotic arm (B. Ekström M. Ronaghi T. Nordström P. Nyrén M. Uhlén unpublished data). The sample preparation robot was based on streptavidin-coated magnetic particles for PCR-capture and handling (A. Holmberg and M. Uhlén unpublished data).

Hultman T., Ståhl S., Hornes E., Uhlén M., Nucleic Acids Res.17, 4937 (1989).

We thank K. Nourizad for very helpful technical assistance and A. Scott for critical review. Supported by grants from PyroSequencing AB and the Swedish Research Council for Engineering Science (TFR).