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| 2. |
- Akhras, Michael, et al.
(författare)
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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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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 2:9, s. e915-
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Tidskriftsartikel (refereegranskat)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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| 3. |
- Akhras, Michael, et al.
(författare)
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PathogenMip Assay : A Multiplex Pathogen Detection Assay
- 2007
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 2:2, s. e223-
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Tidskriftsartikel (refereegranskat)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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| 4. |
- Akhras, Michael S., 1980-
(författare)
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Nucleic Acid Based Pathogen Diagnostics
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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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| 5. |
- Andersson, A. F., et al.
(författare)
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Comparative analysis of human gut microbiota by barcoded pyrosequencing
- 2008
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 3:7
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Tidskriftsartikel (refereegranskat)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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| 6. |
- Andréasson, Hanna, 1975-
(författare)
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Sensitive Forensic DNA Analysis : Application of Pyrosequencing and Real-time PCR Quantification
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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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| 7. |
- Gharizadeh, Baback, et al.
(författare)
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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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Ingår i: International Journal of Antimicrobial Agents. - : Elsevier BV. - 0924-8579 .- 1872-7913. ; 26:6, s. 486-490
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Tidskriftsartikel (refereegranskat)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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| 8. |
- Gharizadeh, Baback, et al.
(författare)
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Methodological improvements of pyrosequencing technology
- 2006
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Ingår i: Journal of Biotechnology. - : Elsevier BV. - 0168-1656 .- 1873-4863. ; 124:3, s. 504-511
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Tidskriftsartikel (refereegranskat)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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| 9. |
- Gharizadeh, Baback, et al.
(författare)
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Sentinel-base DNA genotyping using multiple sequencing primers for high-risk human papillomaviruses
- 2006
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Ingår i: Mol Cell Probes. - : Elsevier BV. ; 20:3-4, s. 230-238
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Tidskriftsartikel (refereegranskat)abstract
- Despite the various technologies in place for genotyping human papillomaviruses (HPV), clinical use and clinical research demand a method that is fast, more reliable and cost-effective. The technology described here represents a breakthrough development in that direction. By combining the method of multiple sequencing primers with DNA sequencing, we have developed a rapid assay for genotyping HPV that relies on the identification of a single, type-specific 'sentinel' base. As described here, the prototype assay has been developed to recognize the 12 most high-risk HPV types (HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59) and is capable of recognizing and simultaneously genotyping multiple HPV co-infections. By providing sequence information on multiple HPV infections, this method eliminates the need for labor- and cost-intensive PCR cloning. These proof-of-concept studies establish the assay to be accurate, reliable, rapid, flexible, and cost-effective, providing evidence of the feasibility this technique for use in clinical settings.
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| 10. |
- Gharizadeh, B., et al.
(författare)
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Type-specific multiple sequencing primers - A novel strategy for reliable and rapid genotyping of human papilloma viruses by pyrosequencing technology
- 2005
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Ingår i: Journal of Molecular Diagnostics. - 1525-1578 .- 1943-7811. ; 7:2, s. 198-205
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 11. |
- Laytragoon-Lewin, Nongnit, et al.
(författare)
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Human papillomavirus (HPV), DNA aberrations and cell cycle progression in anal squamous cell carcinoma patients
- 2007
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Ingår i: Anticancer Research. - 0250-7005 .- 1791-7530. ; 27:6C, s. 4473-4479
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Tidskriftsartikel (refereegranskat)abstract
- Human papillomavirus (HP) infections of the genital tract are sexually transmitted and prevalent worldwide. In this study, the role of HPV in 72 patients with anal squamous cell carcinoma was investigated. Patients and Methods: Polymerase chain reaction (PCR) in combination with in situ hybridization was used to identify HPV-DNA in the patients biopsies. The HPV typing was conducted by pyrosequencing. Cell cycle and DNA content were analysed by cytometry. Results: Ninety percent of the carcinoma biopsies carried high-risk oncogenic HPV in their malignant cells. Eighty-one percent of these demonstrated a single infection with HPV16, 18 or 33 and 19% were double infected with HPV16 and HPV18 Accumulations of viral genes were seen at the necrotic area of the tumours. The HPV genome in the tumour cell influenced significant the host cell cycle progression, but not DNA aberrations. Within these patients, HPV status in the malignant cells was not found to be associated with patient survival time. Conclusion: High-risk oncogenic HPV may play an important role in the initiation of host cell proliferation in anal squamous cell carcinoma. However, infection with HPV may not have any direct influence itself on the clinical outcome of these patients considering the treatments currently available.
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| 12. |
- Lindback, E., et al.
(författare)
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DNA gyrase gene in Neisseria gonorrhoeae as indicator for resistance to ciprofloxacin and species verification
- 2005
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Ingår i: International Journal of STD and AIDS (London). - : SAGE Publications. - 0956-4624 .- 1758-1052. ; 16:2, s. 142-147
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Tidskriftsartikel (refereegranskat)abstract
- We have identified a unique region of eight amino acids in the quinolone resistance-determining region in the gyrA gene in Neisseria gonorrhoeae as an indicator of resistance to fluoroquinolones. We sequenced that region by the Pyrosequencing(TM) technology in 46 N. gonorrhoeae strains and 11 urine samples positive in AMPLICOR(TM) N. gonorrhoeae polymerase chain reaction (Roche Diagnostics), with corresponding isolates of N. gonorrhoeae. The results showed that 28 samples with minimum inhibitory concentration (MIC) of ciprofloxacin > 1 mg/L had mutations in positions 91 and 95. Fifteen samples with MIC 0.125-1.0 mg/L had either one or both of the mutations. The 14 susceptible samples had no mutations. The target region also discriminates N. gonorrhoeae from other species of Neisseria. Our conclusion is that gyrA is an indicator of resistance to ciprofloxacin in N. gonorrhoeae and sequencing by Pyrosequencing technology is a suitable tool for analysis of DNA in urine samples.
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| 17. |
- Takwa, Mohamad, 1980-
(författare)
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Enzymatic Synthesis of Functional Polyesters
- 2008
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Enzymes are successfully employed in the synthesis of different types of polymers. Candida antarctica lipase B is a highly efficient catalyst for the synthesis of polyesters by ring opening polymerization. ω-Pentadecalactone is an interesting lactone due to the unique proprieties of its polymer (poly-pentadecalactone). These polymers have not been applied in any industrial application due to the difficulties to reach them by chemical polymerization. Enzymatically, poly-pentadecalactone macromonomers can be obtained to high conversion. In this investigation we synthesized difunctionalized poly-pentadecalactone with different functional groups. Taking advantage of the selectivity of Candida antarctica lipase B, we introduced different functional end groups. α,ω-Difunctionalized poly-pentadecalactone macromonomers with two thiol ends, two (meth)acrylate ends or with one thiol and one acrylate end were obtained with a high degree of functional ends. We have improved the difunctionalization procedure to a single-step route for the synthesis of α,ω-functionalized poly-pentadecalactones. This procedure has a great potential for industrial applications due to the simplicity of the process and the clean products afforded. Macromonomers with functionalized ends can be used to obtain new polymer architectures with novel proprieties. We also show how the use of enzymes could have some limitations when using an initiator with a cleavable ester bond. 2-Hydroxyethyl methacrylate (HEMA) was used as initiator for the ring opening polymerization (eROP) of ε-caprolactone and ω-pentadecalactone aiming for methacrylate functional polyester. However, the lipase catalyzed not only the ring opening polymerization but also the cleavage of the HEMA moiety resulting in a mixture of polymer products with various end groups. A kinetics study of the eROP and the transesterification processes when using HEMA showed that the transesterification processes occurs at moderate frequency at low monomer concentration, it becomes dominant at longer reaction times. We showed that fully difunctionalized polymers can be obtained when using HEMA as initiator for the eROP of lactones by adding a proper end capper.
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