| 1. |
- Svantesson, Anna, et al.
(författare)
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A mathematical model of the Pyrosequencing reaction system
- 2004
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Ingår i: Biophysical Chemistry. - : Elsevier BV. - 0301-4622 .- 1873-4200. ; 110:02-jan, s. 129-145
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Tidskriftsartikel (refereegranskat)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. |
- Ahmadian, Afshin, et al.
(författare)
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Analysis of the p53 tumor suppressor gene by pyrosequencing
- 2000
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Ingår i: BioTechniques. - : Future Science Ltd. - 0736-6205 .- 1940-9818. ; 28:1, s. 140-
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Tidskriftsartikel (refereegranskat)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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| 3. |
- Ahmadian, Afshin, et al.
(författare)
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Single-nucleotide polymorphism analysis by pyrosequencing
- 2000
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 280:1, s. 103-110
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Tidskriftsartikel (refereegranskat)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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| 4. |
- Ehn, Maria, et al.
(författare)
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Toward pyrosequencing on surface-attached genetic material by use of DNA-binding luciferase fusion proteins
- 2004
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 329:1, s. 11-20
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Tidskriftsartikel (refereegranskat)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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| 5. |
- Eriksson, Jonas, et al.
(författare)
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7-deaza-2 '-deoxyadenosine-5 '-triphosphate as an alternative nucleotide for the pyrosequencing technology
- 2004
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Ingår i: Nucleosides, Nucleotides & Nucleic Acids. - : Marcel Dekker. - 1525-7770 .- 1532-2335. ; 23:10, s. 1583-1594
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Tidskriftsartikel (refereegranskat)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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| 6. |
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| 7. |
- Eriksson, Jonas, et al.
(författare)
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Method for real-time detection of inorganic pyrophosphatase activity
- 2001
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 293:1, s. 67-70
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Tidskriftsartikel (refereegranskat)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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| 8. |
- Eriksson, Jonas, et al.
(författare)
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Pyrosequencing (TM) technology at elevated temperature
- 2004
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 25:1, s. 20-27
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Tidskriftsartikel (refereegranskat)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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| 9. |
- Eriksson, Jonas, et al.
(författare)
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Pyrosequencing trade mark technology at elevated temperature
- 2004
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Ingår i: Electrophoresis. - : Wiley-VCH Verlagsgesellschaft. - 0173-0835 .- 1522-2683. ; 25:1, s. 20-27
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Tidskriftsartikel (refereegranskat)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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| 10. |
- Gambelunghe, G., et al.
(författare)
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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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Ingår i: Human Immunology. - 0198-8859 .- 1879-1166. ; 64:6, s. 629-632
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Tidskriftsartikel (refereegranskat)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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| 11. |
- Gambelunghe, G., et al.
(författare)
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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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Ingår i: European journal of immunogenetics. - : Wiley. - 0960-7420 .- 1365-2370. ; 31:2, s. 73-76
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Tidskriftsartikel (refereegranskat)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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| 12. |
- Garcia, C. A., et al.
(författare)
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Mutation detection by pyrosequencing : sequencing of exons 5-8 of the p53 tumor suppressor gene
- 2000
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Ingår i: Gene. - 0378-1119 .- 1879-0038. ; 253:2, s. 249-257
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Tidskriftsartikel (refereegranskat)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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| 13. |
- Gharizadeh, Baback, et al.
(författare)
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Identification of medically important fungi by the Pyrosequencing (TM) technology
- 2004
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Ingår i: Mycoses. - : Wiley. - 0933-7407 .- 1439-0507. ; 47:1-2, s. 29-33
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Tidskriftsartikel (refereegranskat)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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| 14. |
- Gharizadeh, B., et al.
(författare)
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Improvements in pyrosequencing technology by employing sequenase polymerase
- 2004
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 330:2, s. 272-280
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Tidskriftsartikel (refereegranskat)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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| 15. |
- Gharizadeh, B., et al.
(författare)
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Long-read pyrosequencing using pure 2 '-deoxyadenosine-5 '-O '-(1-thiotriphosphate) Sp-isomer
- 2002
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 301:1, s. 82-90
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Tidskriftsartikel (refereegranskat)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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| 16. |
- Gharizadeh, B., et al.
(författare)
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Multiple group-specific sequencing primers for reliable and rapid DNA sequencing
- 2003
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Ingår i: Molecular and Cellular Probes. - : Elsevier BV. - 0890-8508 .- 1096-1194. ; 17:4, s. 203-210
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Tidskriftsartikel (refereegranskat)abstract
- Pyrosequencing(TM) technology is a bioluminometric DNA sequencing method that employs a cascade of four enzymes to deliver sequence signals. To date this technology has been limited to the sequencing of short stretches of DNA. As an improvement to this technique, we have introduced a bacterial group-specific, multiple sequencing primer approach that circumvents sequencing of less informative semi-conservative regions of the 16S rRNA gene. This new approach is suitable for challenging templates, improving sequence data quality, avoiding sequencing of non-specific amplification products, lessening sequencing time, and moreover, this strategy should open the way for many new applications in the future. The group-specific, multiple sequencing primers can be applied in the Sanger dideoxy sequencing method as well. In addition, we have improved the chemistry of the Pyrosequencing system enabling sequencing of longer stretches of DNA, which allows numerous new applications.
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| 17. |
- Gharizadeh, Baback, et al.
(författare)
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Multiple group-specific sequencing primers for reliable and rapid DNA sequencing
- 2003
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Ingår i: Molecular and Cellular Probes. - 0890-8508 .- 1096-1194. ; 17:4, s. 203-210
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Tidskriftsartikel (refereegranskat)abstract
- Pyrosequencing™ technology is a bioluminometric DNA sequencing method that employs a cascade of four enzymes to deliver sequence signals. To date this technology has been limited to the sequencing of short stretches of DNA. As an improvement to this technique, we have introduced a bacterial group-specific, multiple sequencing primer approach that circumvents sequencing of less informative semi-conservative regions of the 16S rRNA gene. This new approach is suitable for challenging templates, improving sequence data quality, avoiding sequencing of non-specific amplification products, lessening sequencing time, and moreover, this strategy should open the way for many new applications in the future. The group-specific, multiple sequencing primers can be applied in the Sanger dideoxy sequencing method as well. In addition, we have improved the chemistry of the Pyrosequencing system enabling sequencing of longer stretches of DNA, which allows numerous new applications.
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| 18. |
- Gharizadeh, B., et al.
(författare)
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Multiple-primer DNA sequencing method
- 2003
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 24:08-jul, s. 1145-1151
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Tidskriftsartikel (refereegranskat)abstract
- A multiple-primer DNA sequencing approach suitable for genotyping, detection and identification of microorganisms and viruses has been developed. In this new method two or m ore sequencing primers, combined in a pool, are added to a DNA sample of interest. The oligonucleotide that hybridizes to the DNA sample will function as a primer during the subsequent DNA sequencing procedure. This strategy is suited for selective detection and genotyping of relevant microorganisms and samples harboring different DNA targets such as,multiple variant/infected samples as well as unspecific amplification products. This method is used here in a model system for detection and typing of high-risk oncogenic human papilloma viruses (HPVs) in samples containing multiple infections/variants or unspecific amplification products. Type-specific sequencing primers were designed for four of the most oncogenic (high-risk) HPV types (HPV-16, HPV-18, HPV-33, and HPV-45). The primers were combined and added to a sample containing a mixture of one high-risk (16, 18, 33, or 45) and one or two low-risk types. The DNA samples were sequenced by the Pyrosequencing(TM) technology and the Sanger dideoxy sequencing method. Correct genotyping was achieved in all tested combinations. This multiple-sequencing primer approach also improved the sequence data quality for samples containing unspecific amplification products. The new strategy is highly suitable for diagnostic typing of relevant species/genotypes of microorganisms.
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| 19. |
- Gharizadeh, B., et al.
(författare)
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Typing of human papillomavirus by pyrosequencing
- 2001
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Ingår i: Laboratory Investigation. - : Elsevier BV. - 0023-6837 .- 1530-0307. ; 81:5, s. 673-679
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Tidskriftsartikel (refereegranskat)abstract
- The possibility of using a new bioluminometric UNA 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.
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| 20. |
- Gharizadeh, Baback, et al.
(författare)
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Viral and microbial genotyping by a combination of multiplex competitive hybridization and specific extension followed by hybridization to generic tag arrays
- 2003
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 31:22, s. e146-
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Tidskriftsartikel (refereegranskat)abstract
- Detection and identification of microbial pathogens are important for disease diagnosis, treatment and prophylaxis measurements. By introducing an innovative technique, we show a robust, reliable and accurate microarray-based method for identification of microbial pathogens. The technique utilizes a unique combination of multiplex competitive hybridization, which enhances hybridization accuracy of oligonucleotides to the specific target, and apyrase-mediated allele-specific extension, which improves specific extension. As a model system, different clinically relevant human papillomaviruses were selected for this study. The method generated accurate results and proves to be promising for specific and correct microbial and viral typing.
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| 21. |
- Lundeberg, Joakim, et al.
(författare)
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Allele-specific primer extension assay
- 2001
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Patent (populärvet., debatt m.m.)abstract
- The present invention provides methods of allele-specific primer extension useful for detecting mutations and genetic variations. In particular the invention provides a method of detecting a base at a pre-determined position in a nucleic acid molecule, said method comprising: performing primer extension reactions using base-specific detection primers, each primer being specific for a particular base at said predetermined position, and comparing said primer extensions to determine which base is present at said position, wherein said primer extension reactions are performed using labelled nucleotides and wherein a nucleotide-degrading enzyme is present during the primer extension reaction.
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| 22. |
- Mendel-Hartvig, Maritha
(författare)
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Padlock Probes and Rolling Circle Amplification : New Possibilities for Sensitive Gene Detection
- 2002
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- A series of novel methods for detection of known sequence variants in DNA, in particular single nucleotide polymorphism, using padlock probes and rolling circle replication are presented. DNA probes that can be circularized – padlock probes – are ideal for rolling circle replication. Circularized, but not unreacted probes, can generate powerful signal amplification by allowing the reacted probes to template a rolling circle replication (RCR) reaction. However, when hybridized and ligated to a target DNA molecule with no nearby ends, the probes are bound to the target sequence, inhibiting the RCR reaction is. This problem can be solved by generating a branched DNA probe with two 3’ arms such that the probes may be circularized while leaving the second 3’ arm as a primer for the RCR reaction. We describe how T4 DNA ligase can be used for efficient construction of DNA molecules having one 5’ end but two distinct 3’ ends that extend from the 2’ and 3’ carbons of an internal nucleotide. An even stronger approach to circumvent the topological problem that can inhibit RCR is to restriction digest the template downstream of the padlock recognition site. By using Phi 29 DNA polymerase with efficient 3’ exonuclease and strand displacement activity, the template strand can then be used to prime the RCR reaction. The amplified molecule is contiguous with the target DNA, generating an anchored localized signal. The kinetics of the reaction was investigated by following the reaction in real-time using molecular beacon probes. Localized RCR signal were obtained on DNA arrays, allowing detection of as little as 104-105 spotted molecules, of either single- or double-stranded M13 DNA, in a model experiment. We have also established a serial rolling circle amplification procedure. By converting rolling circle products to a second and even third generation of padlock probes the signal was amplified thousand-fold per generation. This procedure provides sufficient sensitivity for detection of single-copy gene sequences in 50 ng of human genomic DNA, and large numbers of probes were amplified in parallel with excellent quantitative resolution.
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| 23. |
- Nordstrom, T., et al.
(författare)
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Direct analysis of single-nucleotide polymorphism on double-stranded DNA by pyrosequencing
- 2000
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Ingår i: Biotechnology and applied biochemistry. - 0885-4513 .- 1470-8744. ; 31, s. 107-112
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Tidskriftsartikel (refereegranskat)abstract
- 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 (GPXI) 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.
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| 24. |
- Nordstrom, T., et al.
(författare)
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Method enabling fast partial sequencing of cDNA clones
- 2001
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 292:2, s. 266-271
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Tidskriftsartikel (refereegranskat)abstract
- Pyrosequencing is a nonelectrophoretic single-tube DNA sequencing method that takes advantage of cooperativity between four enzymes to monitor DNA synthesis. To investigate the feasibility of the recently developed technique for tag sequencing, 64 colonies of a selected cDNA library from human were sequenced by both pyrosequencing and Sanger DNA sequencing. To determine the needed length for finding a unique DNA sequence, 100 sequence tags from human were retrieved from the database and different lengths from each sequence were randomly analyzed. An homology search based on 20 and 30 nucleotides produced 97 and 98% unique hits, respectively. An homology search based on 100 nucleotides could identify all searched genes. Pyrosequencing was employed to produce sequence data for 30 nucleotides. A similar search using BLAST revealed 16 different genes. Forty-six percent of the sequences shared homology with one gene at different positions. Two of the 64 clones had unique sequences. The search results from pyrosequencing were in 100% agreement with conventional DNA sequencing methods. The possibility of using a fully automated pyrosequencer machine for future high-throughput tag sequencing is discussed.
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| 25. |
- Nordstrom, T., et al.
(författare)
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Method enabling pyrosequencing on double-stranded DNA
- 2000
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 282:2, s. 186-193
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Tidskriftsartikel (refereegranskat)abstract
- Pyrosequencing is a new nonelectrophoretic, single-tube DNA sequencing method that takes advantage of co-operativity between four enzymes to monitor DNA synthesis (M. Ronaghi, M. Uhlen, and P. Nyren, Science 281, 363-365). Pyrosequencing has so far only been performed on single-stranded DNA, In this paper different enzymatic strategies for template preparation enabling pyrosequencing on double-stranded DNA were studied. High quality data were obtained with several different enzyme combinations: (i) shrimp alkaline phosphatase and exonuclease I, (ii) calf intestine alkaline phosphatase and exonuclease I, (iii) apyrase and inorganic pyrophosphatase together with exonuclease I, and (iv) apyrase and ATP sulfurylase together with exonuclease I. In many cases, when the polymerase chain reaction was efficient exonuclease I could be omitted. In certain cases, additives such as dimethyl sulfoxide, single-stranded DNA-binding protein, and Klenow DNA polymerase improved the sequence quality. Apyrase was the fastest and most efficient of the three different nucleotide degrading enzymes tested. The data quality obtained on double-stranded DNA was comparable with that on single-stranded DNA. Pyrosequencing data for more than 30 bases could be generated on both long and short templates, as well as on templates with high GC content.
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