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A new sequencing method (Pyrosequencing) has been developed
(1,2,3) based on the combination of four enzymes: (i) polymerase
(bacterial), (ii) sulphurylase (yeast), (iii) luciferase (firefly)
and (iV) apyrase (potato). In this way, the incorporation
of a specific nucleotide can be quantitatively detected as
visible light (see figure). This method, which was developed
in collaboration with Prof Pål Nyren (KTH), allows sequencing
to be done in a rapid and simple manner using a micro titer
plate format (3). Recently, automated systems for analysis
of single nucleotide polymorphisms (SNP) has been developed
consisting of three separate unit operations; (i) PCR, (ii)
sample preparation, including primer annealing and (iii) pyrosequencing,
including SNP-scoring and quality assessment. The system has
been used for analysis of linkage disequilibrium in small
human populations (5) and for pharmacogenetic analysis of
patients treated for various diseases, including cardiovascular
disorders (6).
| Key (own)
publications: |
| 1. |
Nyren, Pettersson & Uhlen
Solid phase DNA minisequencing by an enzymatic luminometric
inorganic pyrophosphate detection assay. Anal Biochem 208,
171-175 (1993). |
| 2. |
Ronaghi, Karamohamed, Pettersson, Uhlen &
Nyren Real-time DNA sequencing using detection of pyrophosphate
release. Anal Biochem 242, 84-89 (1996). |
| 3. |
Ronaghi, Uhlen & Nyren A sequencing method
based on real-time pyrophosphate. Science 281, 363, 365
(1998). |
| 4. |
Ahmadian et al. Single-nucleotide polymorphism
analysis by pyrosequencing. Anal Biochem 280, 103-110 (2000). |
5. |
O’Meara D., Wilbe K., Albert J., and
Lundeberg J. Monitoring resistance to HIV-1 protease inhibitors
by pyrosequencing. 2001, J Clin Micro, 39, 464-473 |
6. |
Gustafsson A., Kiljas J., Alderborn A., Uhlén
M., Andersson L., and Lundeberg J. Screening of single nucleotide
polymorphisms in the pig melanocortin receptor 1 using pyrosequencing
technology. 2002, Animal Biotech, 145-154 |
7. |
Kaessmann H., Zöllner S., Gustafsson
A., Wiebe V., Laan M., Lundeberg J., Uhlen M., and Pääbo
S. Extensive linkage disequilibrium in small human populations
in Eurasia. 2002 Am J of Hum Genet, 70, 673-685 |
8. |
Agaton C., Sievertzon M., Holmberg A., Larsson
M., Unneberg P., Odeberg J., Uhlén M., and Lundeberg
J. Gene expression analysis by signature pyrosequencing.
2002, Gene, 289, 31-39 |
9. |
Ehn M, Ahmadian A., Nilsson P., Lundeberg
J. and Hober S. Esherichia coli Single-stranded DNA-binding
protein (SSB), a molecular tool for improved sequence quality
in pyrosequencing. Electrophoresis, 23:3289-3299 |
10. |
Odeberg J, Holmberg K, Eriksson P, Uhlén
M. Molecular Haplotyping by Pyrosequencing. Biotechniques
2002;33(5): |
11. |
Persson Å, Platz A., Hansson J and
Lundeberg J. Detection of N-ras mutations in melanoma tissue
samples by pyrosequencing, 2002, Clin Chem, in press |
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