| 1. |
- Svantesson, Anna, et al.
(author)
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A mathematical model of the Pyrosequencing reaction system
- 2004
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In: Biophysical Chemistry. - : Elsevier BV. - 0301-4622 .- 1873-4200. ; 110:02-jan, s. 129-145
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Journal article (peer-reviewed)abstract
- The Pyrosequencing(TM) technology is a newly developed DNA sequencing method that monitors DNA nucleotide incorporation in real-time. A set of coupled enzymatic reactions, together with bioluminescence, detects incorporated nucleotides in the form of light pulses, yielding a characteristic light profile. In this study, a biochemical model of the Pyrosequencing reaction system is suggested and implemented. The model is constructed utilizing an assumption of irreversible Michaelis-Menten rate equations and a constant incorporation efficiency. The kinetic parameters are studied and values are chosen to obtain as reliable simulation results as possible. The results presented here show strong resemblance with real experiments. The model is able to capture the dynamics of a single light pulse with great accuracy, as well as the overall characteristics of a whole pyrogram(TM). The plus- and minus-shift effects observed in experiments are successfully reconstructed by two constant efficiency factors. Furthermore, pulse broadening can partly be explained by apyrase inhibition and successive dilution.
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| 2. |
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| 3. |
- Ahmadian, Afshin, et al.
(author)
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Analysis of the p53 tumor suppressor gene by pyrosequencing
- 2000
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In: BioTechniques. - : Future Science Ltd. - 0736-6205 .- 1940-9818. ; 28:1, s. 140-
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Journal article (peer-reviewed)abstract
- Tumor suppressor genes are implicated in cell cycle progression. Inactivation of these genes predominantly occurs through mutations and/or allelic loss that involves both alleles. With inactivation by multiple mutations in a single gene, cloning of the amplified gene is necessary to determine whether the mutations reside on one ol both alleles. Using pyrosequencing, a recently developed approach based on sequencing-by-synthesis, we studied genetic variability in the p53 tumor suppressor gene and could quantify the ratio between the mutated and wild-type amplified fragments. Further-more, this sequencing technique also allows allelic determination of adjacent mutations with no cloning of amplified fragments.
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| 4. |
- Ahmadian, Afshin, et al.
(author)
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Single-nucleotide polymorphism analysis by pyrosequencing
- 2000
-
In: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 280:1, s. 103-110
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Journal article (peer-reviewed)abstract
- There is a growing demand for high-throughput methods for analysis of single-nucleotide polymorphic (SNP) positions. Here, we have evaluated a novel sequencing approach, pyrosequencing, for such purposes. Pyrosequencing is a sequencing-by-synthesis method in which a cascade of enzymatic reactions yields detectable light, which is proportional to incorporated nucleotides. One feature of typing SNPs with pyrosequencing is that each allelic variant will give a unique sequence compared to the two other variants. These variants can easily be distinguished by a pattern recognition software. The software displays the allelic: alternatives and allows for direct comparison with the pyrosequencing raw data. For optimal determination of SNPs, various protocols of nucleotide dispensing order were investigated. Here, we demonstrate that typing of SNPs can efficiently be performed by pyrosequencing using an automated system for parallel analysis of 96 samples in approximately 5 min, suitable for large-scale screening and typing of SNPs.
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| 5. |
- Akhras, Michael, et al.
(author)
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Connector Inversion Probe Technology : A Powerful One- Primer Multiplex DNA Amplification System for Numerous Scientific Applications
- 2007
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In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 2:9, s. e915-
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Journal article (peer-reviewed)abstract
- We combined components of a previous assay referred to as Molecular Inversion Probe (MIP) with a complete gap filling strategy, creating a versatile powerful one-primer multiplex amplification system. As a proof-of-concept, this novel method, which employs a Connector Inversion Probe (CIPer), was tested as a genetic tool for pathogen diagnosis, typing, and antibiotic resistance screening with two distinct systems: i) a conserved sequence primer system for genotyping Human Papillomavirus (HPV), a cancer-associated viral agent and ii) screening for antibiotic resistance mutations in the bacterial pathogen Neisseria gonorrhoeae. We also discuss future applications and advances of the CIPer technology such as integration with digital amplification and next-generation sequencing methods. Furthermore, we introduce the concept of two-dimension informational barcodes, i. e. "multiplex multiplexing padlocks'' (MMPs). For the readers' convenience, we also provide an on-line tutorial with user-interface software application CIP creator 1.0.1, for custom probe generation from virtually any new or established primer-pairs.
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| 6. |
- Akhras, Michael, et al.
(author)
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PathogenMip Assay : A Multiplex Pathogen Detection Assay
- 2007
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In: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 2:2, s. e223-
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Journal article (peer-reviewed)abstract
- The Molecular Inversion Probe (MIP) assay has been previously applied to a large-scale human SNP detection. Here we describe the PathogenMip Assay, a complete protocol for probe production and applied approaches to pathogen detection. We have demonstrated the utility of this assay with an initial set of 24 probes targeting the most clinically relevant HPV genotypes associated with cervical cancer progression. Probe construction was based on a novel, cost-effective, ligase-based protocol. The assay was validated by performing pyrosequencing and Microarray chip detection in parallel experiments. HPV plasmids were used to validate sensitivity and selectivity of the assay. In addition, 20 genomic DNA extracts from primary tumors were genotyped with the PathogenMip Assay results and were in 100% agreement with conventional sequencing using an L1-based HPV genotyping protocol. The PathogenMip Assay is a widely accessible protocol for producing and using highly discriminating probes, with experimentally validated results in pathogen genotyping, which could potentially be applied to the detection and characterization of any microbe.
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| 7. |
- Akhras, Michael S., 1980-
(author)
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Nucleic Acid Based Pathogen Diagnostics
- 2008
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Doctoral thesis (other academic/artistic)abstract
- Pathogenic organisms are transmitted to the host organism through all possible connected pathways, and cause a myriad of diseases states. Commonly occurring curable infectious diseases still impose the greatest health impacts on a worldwide perspective. The Bill & Melinda Gates Foundation partnered with RAND Corporation to form the Global Health Diagnostics Forum, with the goal of establishing and interpreting mathematical models for what effects a newly introduced point-of-care pathogen diagnostic would have in developing countries. The results were astonishing, with potentially millions of lives to be saved on an annual basis. Golden standard for diagnostics of pathogenic bacteria has long been cultureable medias. Environmental biologists have estimated that less than 1% of all bacteria are cultureable. Genomic-based approaches offer the potential to identify all microbes from all the biological kingdoms. Nucleic acid based pathogen diagnostics has evolved significantly over the past decades. Novel technologies offer increased potential in sensitivity, specificity, decreased costs and parallel sample management. However, most methods are confined to core laboratory facilities. To construct an ultimate nucleic acid based diagnostic for use in areas of need, potential frontline techniques need to be identified and combined. The research focus of this doctoral thesis work has been to develop and apply nucleic acid based methods for pathogen diagnostics. Methods and assays were applied to the two distinct systems i) screening for antibiotic resistance mutations in the bacterial pathogen Neisseria gonorrhoeae, and ii) genotype determination of the cancer causative Human Papillomavirus (HPV). The first part of the study included development of rapid, direct and multiplex Pyrosequencing nucleic acid screenings. With improved methodology in the sample preparation process, we could detect an existence of multiple co-infecting HPV genotypes at greater sensitivities than previously described, when using the same type of methodology. The second part of the study focused on multiplex nucleic acid amplification strategies using Molecular Inversion Probes with end-step Pyrosequencing screening. The PathogenMip assay presents a complete detection schematic for virtually any known pathogenic organism. We also introduce the novel Connector Inversion Probe, a padlock probe capable of complete gap-fill reactions for multiplex nucleic acid amplifications.
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| 8. |
- Andersson, A. F., et al.
(author)
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Comparative analysis of human gut microbiota by barcoded pyrosequencing
- 2008
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In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 3:7
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Journal article (peer-reviewed)abstract
- 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.
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| 9. |
- Andréasson, Hanna, 1975-
(author)
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Sensitive Forensic DNA Analysis : Application of Pyrosequencing and Real-time PCR Quantification
- 2005
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Doctoral thesis (other academic/artistic)abstract
- The field of forensic genetics is growing fast and the development and optimisation of more sensitive, faster and more discriminating forensic DNA analysis methods is highly important. In this thesis, an evaluation of the use of novel DNA technologies and the development of specific applications for use in forensic casework investigations are presented.In order to maximise the use of valuable limited DNA samples, a fast and user-friendly Real-time PCR quantification assay, of nuclear and mitochondrial DNA copies, was developed. The system is based on the 5’ exonuclease detection assay and was evaluated and successfully used for quantification of a number of different evidence material types commonly found on crime scenes. Furthermore, a system is described that allows both nuclear DNA quantification and sex determination in limited samples, based on intercalation of the SYBR Green dye to double stranded DNA. To enable highly sensitive DNA analysis, Pyrosequencing of short stretches of mitochondrial DNA was developed. The system covers both control region and coding region variation, thus providing increased discrimination power for mitochondrial DNA analysis. Finally, due to the lack of optimal assays for quantification of mitochondrial DNA mixture, an alternative use of the Pyrosequencing system was developed. This assay allows precise ratio quantification of mitochondrial DNA in samples showing contribution from more than one individual.In conclusion, the development of optimised forensic DNA analysis methods in this thesis provides several novel quantification assays and increased knowledge of typical DNA amounts in various forensic samples. The new, fast and sensitive mitochondrial DNA Pyrosequencing assay was developed and has the potential for increased discrimination power.
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| 10. |
- Babrzadeh, F., et al.
(author)
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Collinearity of protease mutations in HIV-1 samples with high-level protease inhibitor class resistance
- 2013
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In: Journal of Antimicrobial Chemotherapy. - : Oxford University Press (OUP). - 0305-7453 .- 1460-2091. ; 68:2, s. 414-418
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Journal article (peer-reviewed)abstract
- Objectives: To determine whether pan-protease inhibitor (PI)-resistant virus populations are composed predominantly of viruses with resistance to all PIs or of diverse virus populations with resistance to different subsets of PIs. Methods: We performed deep sequencing of plasma virus samples from nine patients with high-level genotypic and/or phenotypic resistance to all licensed PIs. The nine virus samples had a median of 12 PI resistance mutations by direct PCR Sanger sequencing. Results: For each of the nine virus samples, deep sequencing showed that each of the individual viruses within a sample contained nearly all of the mutations detected by Sanger sequencing. Indeed, a median of 94.9% of deep sequence reads had each of the PI resistance mutations present as a single chromatographic peak in the Sanger sequence. A median of 5.0% of reads had all but one of the Sanger mutations that were not part of an electrophoretic mixture. Conclusions: The collinearity of PI resistance mutations in the nine virus samples demonstrated that pan-PI-resistant viruses are able to replicate in vivo despite their highly mutated protease enzymes. We hypothesize that the marked collinearity of PI resistance mutations in pan-PI-resistant virus populations results from the unique requirements for multi-PI resistance and the extensive cross-resistance conferred by many of the accessory PI resistance mutations.
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| 11. |
- Babrzadeh, Farbod, et al.
(author)
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Whole-genome sequencing of the efficient industrial fuel-ethanol fermentative Saccharomyces cerevisiae strain CAT-1
- 2012
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In: Molecular Genetics and Genomics. - : Springer Science and Business Media LLC. - 1617-4615 .- 1617-4623. ; 287:6, s. 485-494
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Journal article (peer-reviewed)abstract
- The Saccharomyces cerevisiae strains widely used for industrial fuel-ethanol production have been developed by selection, but their underlying beneficial genetic polymorphisms remain unknown. Here, we report the draft whole-genome sequence of the S. cerevisiae strain CAT-1, which is a dominant fuel-ethanol fermentative strain from the sugarcane industry in Brazil. Our results indicate that strain CAT-1 is a highly heterozygous diploid yeast strain, and the similar to 12-Mb genome of CAT-1, when compared with the reference S228c genome, contains similar to 36,000 homozygous and similar to 30,000 heterozygous single nucleotide polymorphisms, exhibiting an uneven distribution among chromosomes due to large genomic regions of loss of heterozygosity (LOH). In total, 58 % of the 6,652 predicted protein-coding genes of the CAT-1 genome constitute different alleles when compared with the genes present in the reference S288c genome. The CAT-1 genome contains a reduced number of transposable elements, as well as several gene deletions and duplications, especially at telomeric regions, some correlated with several of the physiological characteristics of this industrial fuel-ethanol strain. Phylogenetic analyses revealed that some genes were likely associated with traits important for bioethanol production. Identifying and characterizing the allelic variations controlling traits relevant to industrial fermentation should provide the basis for a forward genetics approach for developing better fermenting yeast strains.
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| 12. |
- Ehn, Maria, et al.
(author)
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Toward pyrosequencing on surface-attached genetic material by use of DNA-binding luciferase fusion proteins
- 2004
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In: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 329:1, s. 11-20
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Journal article (peer-reviewed)abstract
- Mutation detection and single-nucleotide polymorphisin genotyping require screening of large samples of materials and therefore the importance of high-throughput DNA analysis techniques is significant. Pyrosequencing is a four-enzyme bioluminometric DNA sequencing technology based on the sequencing-by-synthesis principle. Currently, the technique is limited to simultaneous analysis of 96 or 384 samples. Earlier, attempts to increase the sample capacity were made using micromachined filter chamber arrays where parallel analyses of nanoliter samples could be monitored in real time. We have developed a strategy for specific immobilization of the light-producing enzyme luciferase to the DNA template within a reaction chamber. By this approach, luciferase is genetically fused to a DNA-binding protein (Klenow polymerase or Escherichia coli single-stranded DNA-binding (SSB) protein) and to a purification handle (Z(basic)). The proteins are produced in E. coli and purified using cation and anion exchange chromatography with removal of Z(basic). The produced proteins have been analyzed using an assay for complete primer extension of DNA templates immobilized on magnetic beads detected by pyrosequencing chemistry. Results from these experiments show that the proteins bind selectively to the immobilized DNA and that their enzymatic domains were active. Z(basic)-SSB-luciferase produced the highest signal in this assay and was further exploited as enzymatic reagent for DNA sequencing.
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| 13. |
- Eriksson, Jonas, et al.
(author)
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7-deaza-2 '-deoxyadenosine-5 '-triphosphate as an alternative nucleotide for the pyrosequencing technology
- 2004
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In: Nucleosides, Nucleotides & Nucleic Acids. - : Marcel Dekker. - 1525-7770 .- 1532-2335. ; 23:10, s. 1583-1594
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Journal article (peer-reviewed)abstract
- A new adenosine nucleotide analog suitable for the Pyrosequencing method is presented. The new analog, 7-deaza-2'-deoxyadenosine-5'-triphosphate (c(7)dATP), has virtually the. same low substrate specificity for luciferase as the currently used analog, 2'-deoxyadenosine-5'-O-(1-thiotriphosphate) (dATPalphaS). The inhibitory effect dATPalphaS displays on the nucleotide degrading activity of apyrase was reduced significantly by substituting the c(7)dATP for the dATPalphaS. Both analogs show high stability after long time storage at +8degreesC. Furthermore, with the new nucleotide a read length of up to 100 bases was obtained for several templates from fungi, bacteria and viruses.
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| 14. |
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| 15. |
- Eriksson, Jonas, et al.
(author)
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Method for real-time detection of inorganic pyrophosphatase activity
- 2001
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In: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 293:1, s. 67-70
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Journal article (peer-reviewed)abstract
- A sensitive and simple method for real-time detection of inorganic pyrophosphatase (PPase) (EC 3.6.1.1) activity has been developed. The method is based on PPase-induced activation of the firefly luciferase activity in the presence of inorganic pyrophosphate (PPi). PPi inhibits the luciferase activity, but in the presence of PPase the luciferase activity is restored and the luminescence output increases. The assay yields linear responses between 8 and 500 mU. The detection limit was found to be 8 mU PPase. The method was used to detect the hydrolytic activity of PPases from Saccharomyces cerevisiae, Escherichia coli, and Bacillus stearothermophilus. As substrate for the luciferase, adenosine 5'-phosphosulfate can replace ATP, which is an advantage for detection of PPase activity in crude extracts containing ATP-hydrolyzing activities. The method can be used for kinetic and inhibition studies as well as for detection of PPase activity during different purification procedures.
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| 16. |
- Eriksson, Jonas, et al.
(author)
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Pyrosequencing (TM) technology at elevated temperature
- 2004
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In: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 25:1, s. 20-27
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Journal article (peer-reviewed)abstract
- To date, the Pyrosequencing(TM) technology has been performed at 28degreesC due to the low thermostability of the firefly luciferase. In this study, firefly luciferase was stabilized in the presence of glycine betaine, allowing DNA sequencing at 37degreesC. By increasing the temperature to 37degreesC, false signals due to primer-dimers and loop-structures were decreased significantly. In addition, a combination of (i) replacing the natural dGTP with 7'deaza-dGTP in the polymerase chain reaction (PCR), (ii) 1.6 m glycine betaine, and (iii) an increase of the temperature to 37degreesC enabled us to sequence a DNA template with the initial sequence 3'-ATGGCCCGGGGGGGAGCTCCA . . . 5'. Furthermore, we describe a method to analyze if a primer forms a primer-dimer with extendable 3'-ends.
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| 17. |
- Eriksson, Jonas, et al.
(author)
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Pyrosequencing trade mark technology at elevated temperature
- 2004
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In: Electrophoresis. - : Wiley-VCH Verlagsgesellschaft. - 0173-0835 .- 1522-2683. ; 25:1, s. 20-27
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Journal article (peer-reviewed)abstract
- To date, the Pyrosequencing trade mark technology has been performed at 28 degrees C due to the low thermostability of the firefly luciferase. In this study, firefly luciferase was stabilized in the presence of glycine betaine, allowing DNA sequencing at 37 degrees C. By increasing the temperature to 37 degrees C, false signals due to primer-dimers and loop-structures were decreased significantly. In addition, a combination of (i) replacing the natural dGTP with 7'deaza-dGTP in the polymerase chain reaction (PCR), (ii) 1.6 M glycine betaine, and (iii) an increase of the temperature to 37 degrees C enabled us to sequence a DNA template with the initial sequence 3'-ATGGCCCGGGGGGGAGCTCCA em leader 5'. Furthermore, we describe a method to analyze if a primer forms a primer-dimer with extendable 3'-ends.
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| 18. |
- Gambelunghe, G., et al.
(author)
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Lack of association of CCR2-64I and CCR5-Delta 32 with type 1 diabetes and latent autoimmune diabetes in adults
- 2003
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In: Human Immunology. - 0198-8859 .- 1879-1166. ; 64:6, s. 629-632
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Journal article (peer-reviewed)abstract
- It is well known that type I diabetes mellitus (T1DM) is a complex genetic disease resulting from the autoimmune destruction of pancreatic beta cells. Several genes have been associated with susceptibility and/or protection for T1DM, but the disease risk is mostly influenced by genes located in the class II region of the major histocompatibility complex. The attraction of leukocytes to tissues is essential for inflammation and the beginning of autoimmune reaction. The process is controlled by chemokines, which are chemotactic cytolines. Some studies have shown that CCR2-64I and CCR5-Delta32 might be important for protection of susceptibility to some immunologically-mediated disorders. In the present study, we demonstrate the lack of association between CCR2-64I and CCR5-Delta32 gene polymorphism and TIDM and we desrcibe a new method for a simple and more precise genotyping of the CCR2 gene.
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| 19. |
- Gambelunghe, G., et al.
(author)
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Lack of association of human chemokine receptor gene polymorphisms CCR2-64I and CCR5-Delta 32 with autoimmune Addison's disease
- 2004
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In: European journal of immunogenetics. - : Wiley. - 0960-7420 .- 1365-2370. ; 31:2, s. 73-76
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Journal article (peer-reviewed)abstract
- The attraction of leukocytes to tissues is essential for inflammation and the initiation of the autoimmune reaction. The process is controlled by chemokines, which are chemotactic cytokines. We investigated whether human chemokine receptor gene polymorphisms, namely CCR5-Delta32 and CCR2-64I, are associated with susceptibility to autoimmune Addison's disease. Genotyping was performed in 56 patients and 127 healthy controls by a new method using pyrosequencing for CCR2-64I and by polymerase chain reaction and detecting gel for CCR5-Delta32. None of the CCR2 or CCR5 alleles was found to be associated, either positively or negatively, with disease risk. Our results indicate that the CCR2-64I and CCR5-Delta32 gene polymorphisms do not play a major role in conferring genetic risk for, and/or protection against, autoimmune Addison's disease.
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| 20. |
- Garcia, C. A., et al.
(author)
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Mutation detection by pyrosequencing : sequencing of exons 5-8 of the p53 tumor suppressor gene
- 2000
-
In: Gene. - 0378-1119 .- 1879-0038. ; 253:2, s. 249-257
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Journal article (peer-reviewed)abstract
- 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(TM) 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.
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| 21. |
- Gharizadeh, Baback, et al.
(author)
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Detection of gyrA mutations associated with ciprofloxacin resistance in Neisseria gonorrhoeae by rapid and reliable pre-programmed short DNA sequencing
- 2005
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In: International Journal of Antimicrobial Agents. - : Elsevier BV. - 0924-8579 .- 1872-7913. ; 26:6, s. 486-490
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Journal article (peer-reviewed)abstract
- Quinolone resistance is rapidly increasing in Neisseria gonorrhoeae and is posing a significant public health threat that requires constant surveillance. A rapid and reliable mutation detection assay has been developed. The assay is based on pre-programmed short DNA sequencing and is designed to detect point mutations in the gyrA gene that are highly related to ciprofloxacin resistance, i.e. in codons 91 and 95. By developing an assay based on pyrosequencing and exploiting the pre-programmed nucleotide dispensation capability of this technology, the sequence comprising the mutations will be analysed and promptly reveal whether the N. gonorrhoeae pathogen carries resistance to ciprofloxacin. A panel of 40 N. gonorrhoeae clinical isolates, of which 27 phenotypically displayed decreased susceptibility or resistance to ciprofloxacin, was used in the present study. All point mutations in the short stretch of the N. gonorrhoeae gyrA gene were easily discriminated, and the genotypic results obtained by pre-programmed sequencing were mainly in agreement with the phenotypically identified decreased susceptibility or resistance to ciprofloxacin. The new method used in the present study has the potential for rapid and reliable identification of known as well as previously unknown drug resistance mutations.
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| 22. |
- Gharizadeh, Baback, et al.
(author)
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Identification of medically important fungi by the Pyrosequencing (TM) technology
- 2004
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In: Mycoses. - : Wiley. - 0933-7407 .- 1439-0507. ; 47:1-2, s. 29-33
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Journal article (peer-reviewed)abstract
- The Pyrosequencing(TM) 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.
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| 23. |
- Gharizadeh, B., et al.
(author)
-
Improvements in pyrosequencing technology by employing sequenase polymerase
- 2004
-
In: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 330:2, s. 272-280
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Journal article (peer-reviewed)abstract
- Pyrosequencing is a DNA sequencing technique based on the bioluminometric detection of inorganic pyrophosphate, which is released when nucleotides are incorporated into a target DNA. Since the technique is based on an enzymatic cascade, the choice of enzymes is a critical factor for efficient performance of the sequencing reaction. In this study we have analyzed the performance of an alternative DNA polymerase, Sequenase, on the sequencing performance of the Pyrosequencing technology. Compared to the Klenow fragment of DNA polymerase I, Sequenase could read through homopolymeric regions with more than five T bases. In addition, Sequenase reduces remarkably interference from primer-dimers and loop structures that give rise to false sequence signals. By using Sequenase, synchronized extensions and longer reads can be obtained on challenging templates, thereby opening new avenues for applications of Pyrosequencing technology.
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| 24. |
- Gharizadeh, B., et al.
(author)
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Long-read pyrosequencing using pure 2 '-deoxyadenosine-5 '-O '-(1-thiotriphosphate) Sp-isomer
- 2002
-
In: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 301:1, s. 82-90
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Journal article (peer-reviewed)abstract
- Pyrosequencing, a nonelectrophoretic DNA sequencing method that uses a luciferase-based enzymatic system to monitor DNA synthesis in real time, has so far been limited to sequencing of short stretches of DNA. To increase the signal-to-noise ratio in pyrosequencing the natural dATP was replaced by dATPalphaS (M. Ronaghi et al., 1996, Anal. Biochem. 242, 84-89). The applied dATPaS was a mixture of two isomers (Sp and Rp). We show here that by the introduction of pure 2'-deoxyadenosine-5'-O'-(1-thiotriphosphate) Sp-isomer in pyrosequencing substantial longer reads could be obtained. The pure Sp-isomer allowed lower nucleotide concentration to be used and improved the possibility to read through poly(T) regions. In general, a doubling of the read length could be obtained by the use of pure Sp-isomer. Pyrosequencing data for 50 to 100 bases could be generated on different types of template. The longer read will enable numerous new applications, such as identification and typing of medically important microorganisms as well as resequencing of DNA fragments for mutation screening and clone checking.
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| 25. |
- Gharizadeh, Baback, et al.
(author)
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Methodological improvements of pyrosequencing technology
- 2006
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In: Journal of Biotechnology. - : Elsevier BV. - 0168-1656 .- 1873-4863. ; 124:3, s. 504-511
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Journal article (peer-reviewed)abstract
- Pyrosequencing technology is a rather novel DNA sequencing method based on the sequencing-by-synthesis principle. This bioluminometric, real-time DNA sequencing technique employs a cascade of four enzymatic reactions producing sequence peak signals. The method has been proven highly suitable for single nucleotide polymorphism analysis and sequencing of short stretches of DNA. Although the pyrosequencing procedure is relatively straightforward, users may face challenges due to varying parameters in PCR and sequencing primer design, sample preparation and nucleotide dispensation; such challenges are labor and cost intensive. In this study, these issues have been addressed to increase signal quality and assure sequence accuracy.
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