Pål Nyrén

Comparative Analysis of Human Gut Microbiota by Barcoded Pyrosequencing

Humans host complex microbial communities believed to contribute to health maintenance and, when in imbalance, to the development of diseases. Determining the microbial composition in patients and healthy controls may thus provide novel therapeutic targets. For this purpose, high-throughput, cost-effective methods for microbiota characterization are needed. We have employed 454-pyrosequencing of a hyper-variable region of the 16S rRNA gene in combination with sample-specific barcode sequences which enables parallel in-depth analysis of hundreds of samples with limited sample processing. In silico modeling demonstrated that the method correctly describes microbial communities down to phylotypes below the genus level. Here we applied the technique to analyze microbial communities in throat, stomach and fecal samples. Our results demonstrate the applicability of barcoded pyrosequencing as a high-throughput method for comparative microbial ecology.

Enzymatic method for continuous monitoring of DNA polymerase activity

A simple and rapid method for the assay of DNA polymerase activity has been developed. The PPi formation in the DNA polymerase reaction is continuously monitored by a coupled enzymatic method (P. Nyrén and A. Lundin, 1985, Anal. Biochem. 151, 504-509) utilizing the enzymes ATP-sulfurylase and firefly luciferase. The method has been used for continuous monitoring of DNA synthesis in vitro, and the effect of an inhibitor, adriamycin, on the polymerase activity was studied. The assay is very sensitive and yields linear responses between 1.5 and 30 micrograms/ml of DNA polymerase (Micrococcus luteus) (2-40 pmol of PPi generated per minute).

Mutation detection by pyrosequencing : sequencing of exons 5-8 of the p53 tumor suppressor gene

The ability to sequence a large number of DNA samples rapidly and accurately for detection of all possible mutations is a critical goal for the future application of DNA sequencing in routine medical diagnostics. Pyrosequencing() is a non-electrophoretic real-time DNA sequencing method that uses the luciferase-luciferin light release as the detection signal for nucleotide incorporation into target DNA. For pyrosequencing of the human p53 gene, a nested multiplex PCR method for amplification of exons 5-8 was prepared. In order to investigate the use of pyrosequencing in mutation detection, DNA samples from skin-cancer patients were used. Two forms of nucleotide dispensation strategy were used, cyclic and programmed. Bi-directional pyrosequencing was performed and the overlapping sequence data produced were assembled to determine the sequence of the gene. Reliable sequencing data were obtained with both dispensation strategies, but some advantages were obtained using the programmed nucleotide dispensation approach, such as longer and faster reads, and fewer out-of-phase problems. The accuracy of pyrosequencing for detection of p53 mutations and allele distribution was demonstrated.

Typing of Human Papillomavirus by Pyrosequencing

The possibility of using a new bioluminometric DNA sequencing technique, called pyrosequencing, for typing of human papillomaviruses (HPV) was investigated. A blinded pyrosequencing test was performed on an HPV test panel of 67 GP5+/GP6+ PCR-derived amplification products. The 67 clinical DNA samples were sequenced up to 25 bases and sequences were searched using BLAST. All of the samples were correctly genotyped by pyrosequencing and the results were unequivocally in accordance with the results obtained from conventional DNA sequencing. Pyrosequencing was found to be a fast and efficient tool for identifying individual HPV types. Furthermore, pyrosequencing has the capability of determining novel HPV types as well as HPV sequence variants harboring mutation(s). The method is robust and well suited for large-scale programs.

Direct analysis of single‐nucleotide polymorphism on double‐stranded DNA by pyrosequencing

Pyrosequencing, a new method for DNA sequencing, is gaining widespread use for many different types of DNA analysis. The method takes advantage of four coupled enzymes in a single tube assay to monitor DNA synthesis in real time using a luminometric detection system. Here, we demonstrate the use of pyrosequencing for direct analysis of single‐nucleotide polymorphism on double‐stranded PCR product. Pyrosequencing data on the human glutathione peroxidase gene (GPX1) from several individuals were analysed and three different allelic variants were determined and confirmed. The possibility of further simplifying the sequencing and template‐preparation steps is discussed.

Identification of medically important fungi by the Pyrosequencing technology.

The PyrosequencingTM technology was used for identification of different clinically relevant fungi. The tests were performed on amplicons derived from the 18S rRNA gene using polymerase chain reaction (PCR) universal primers for amplification. Sequencing was performed up to 40 bases in a variable region with a designed general sequencing primer and the Pyrosequence data were analyzed by BLAST sequence search in the GenBank database. DNA from a total of 21 fungal specimens consisting of nine strains of clinically relevant fungi and 12 clinical specimens from patients suffering from proven invasive fungal infections were PCR‐amplified and analyzed by gel electrophoresis, PCR‐enzyme‐linked immunosorbent assay (ELISA) and the Pyrosequencing technology. All data obtained by the Pyrosequencing technology were in agreement with the results obtained by PCR‐ELISA using species/genus‐specific oligonucleotides and were as well in accordance with the culture results. The results demonstrate that the Pyrosequencing method is a reproducible and reliable technique for identification of fungal pathogens.

Type-specific multiple sequencing primers: a novel strategy for reliable and rapid genotyping of human papillomaviruses by pyrosequencing technology.

DNA sequencing is the gold standard method for accurate microbial and viral typing. However, DNA sequencing techniques have been facing limitations in typing of human papillomaviruses when the specimen harbors multiple genotypes and yields nonspecific amplification products, resulting in nonspecific and noninterpretable sequence data. To address these limitations we have developed a type-specific multiple sequencing primer DNA-sequencing method. This new strategy is suitable for sequencing and typing of samples harboring different genotypes (co-infections with multiple genotypes) and yielding nonspecific amplifications, thus eliminating the need for nested polymerase chain reaction (PCR), stringent PCR conditions, and cloning. The new approach has also proved useful for amplicons containing low PCR yield or subdominant types, avoiding reperforming of amplifications. We have applied the multiple sequencing primer method for genotyping of clinically relevant human papillomaviruses in a clinical test panel by using a combined pool of seven type-specific sequencing primers for HPV-6, -11, -16, -18, -31, -33, and -45. Furthermore, we introduced a sequence pattern recognition approach when there was a plurality of genotypes in the sample to facilitate typing of more than one target DNA in the sample. The multiple sequencing primer method has proved to be a multifaceted approach for typing of human papillomaviruses by DNA sequencing technologies.

Reconstitution of highly purified proton-translocating pyrophosphatase from Rhodospirillum rubrum

Membrane-bound PPase is of interest as it functions in various organisms as a coupling factor between electron transport and PPi synthesis and is involved in the energy transduction pathway as an independent alternative to the ATPase system [1-4]. PPi hydrolysis in chromatophores of Rhodospirillum rubrum caused a change in the fluorescence of added 8-anilino-naphtalene-l-sulfonic acid [5], an uptake of phenyl dicarbaundecaborane anion [6] and a pH change of the outer medium [7]. Therefore, it was concluded that chromatophore PPase translocates protons across the membrane coupled to PPi hydrolysis, apparently in a manner corresponding to that of the chromatophore ATPase. Further investigation of membrane-bound PPase has been impeded by the lack of an efficient method of isolation. Although a method of purifying the PPase has been reported [8], only recently has a method of obtaining a highly purified mem-

Studies on photosynthetic inorganic pyrophosphate formation in Rhodospirillum rubrum chromatophores

Photosynthetic formation of inorganic pyrophosphate (PP(i)) in Rhodospirillum rubrum chromatophores has been studied utilizing a new and sensitive method for continuous monitoring of PP(i) synthesis. Studies of the reaction kinetics under a variety of conditions, e.g., at different substrate concentrations and different electron-transport rates, have been performed. At very low light intensities the rate of PP(i) synthesis is twice the rate of ATP synthesis. Antimycin A, at a concentration which strongly inhibited the photosynthetic ATP formation, inhibited the PP(i) synthesis much less. Even at low rates of electron transport a significant rate of PP(i) synthesis is obtained. The rate of photosynthetic ATP formation is stimulated up to 20% when PPI synthesis is inhibited. It is shown that PP(i) synthesis and ATP synthesis compete with each other. No inhibition of pyrophosphatase activity is observed at high carbonyl cyanide p-trifluoromethoxyhydrazone concentration while ATPase activity is strongly inhibited under the same conditions.

Inorganic pyrophosphate-driven ATP-synthesis in liposomes containing membrane-bound inorganic pyrophosphatase and F0-F1 complex from Rhodospirillum rubrum

PP1 driven ATP synthesis has been reconstituted in a liposomal system containing the membrane‐bound energy‐linked PP1ase and coupling factor complex, both highly purified from Rhodospirillum rubrum. This energy converting model system was made by mixing both enzyme preparations with an aqueous suspension of sonicated soybean phospholipids and subjecting to a freeze—thaw procedure. In the presence of ADP, Mg2+, P1 and PP1 the system catalyzed phosphorylation by up to 25 nmol ATP formed X mg protein−1 X min−1, at 20°C, which was sensitive to uncouplers and inhibitors of phosphorylation such as oligomycin, efrapeptin and N,N′‐dicyclohexylcarbodiimide.