| 1. |
- Ahmadian, Afshin, et al.
(författare)
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A brief history of genetic variation analysis
- 2002
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Ingår i: BioTechniques. - 0736-6205 .- 1940-9818. ; 32:5, s. 1122-
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Forskningsöversikt (refereegranskat)abstract
- As the human genome sequence is determined, there is an emerging need for the analysis of human sequence variations as genetic markers in diagnosis, linkage and association studies, cancer research, and pharmacogenomics. There are several different techniques and approaches for detecting these genetic variations, and here we review some of these techniques and their application fields. However, all the techniques have advantages and disadvantages, and factors such as laboratory instrumentation, personnel experience, required accuracy, required throughput, and cost often have to be taken into account before selecting a method.
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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, 1964-
(författare)
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Approaches for analysis of mutations and genetic variations
- 2001
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Detecting mutations and genomic variations is fundamental indiagnosis, isolating disease genes, association studies,functional genomics and pharmacogenomics. The objective hasbeen to use and further develop a variety of tools andtechnologies to analyze these genetic alterations andvariations. The p53 tumor suppressor gene and short arm of chromosome 9have been used as genetic markers to investigate fundamentalquestions concerning early events preceding non-melanoma skincancers, clonal progression and timing of different mutationsand deletions. Conventional gel based DNA sequencing andfragment analysis of microsatellite markers were utilized forthis purpose. In addition, a sequence-specific PCR-mediatedartifact is discussed. Pyrosequencing, a bioluminometric technique based onsequencing-by-synthesis, has been utilized to determinemutation ratios in the p53 gene. In addition, in the case ofmultiple mutations, pyrosequencing was adopted to determineallelic distribution of mutations without the use of cloningprocedures. Exons 5 to 8 of the p53 gene were also sequenced bythis method. The possibility of typing single base variations bypyrosequencing has been evaluated. Two different nucleotidedispensation orders were investigated and data were comparedwith the predicted pattern for each alternative of the variableposition. Analysis of loss of heterozygosity was possible byutilizing single nucleotide polymorphisms. A modified allele-specific extension strategy for genotypingof single nucleotide polymorphisms has been developed. Throughthe use of a real-time bioluminometric assay, it has beendemonstrated that reaction kinetics for a mismatchedprimer-template is slower than the matched configuration,butthe end-point signals are comparable. By introduction ofapyrase, the problems associated with mismatch extensions havebeen circumvented and accurate data has been obtained. Keywords:fragment analysis, microsatellite, loss ofheterozygosity, DNA sequencing, pyrosequencing, cancer,mutation, variation, single nucleotide polymorphism,allele-specific extension, bioluminescence, apyrase.
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| 4. |
- Ahmadian, Afshin, et al.
(författare)
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Genetic instability in the 9q22.3 region is a late event in the development of squamous cell carcinoma.
- 1998
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Ingår i: Oncogene. - : Springer Science and Business Media LLC. - 0950-9232 .- 1476-5594. ; 17:14
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Tidskriftsartikel (refereegranskat)abstract
- Squamous cell carcinoma (SCC) of the skin represents a group of neoplasms which is associated with exposure to UV light. Recently, we obtained data suggesting that invasive skin cancer and its precursors derive from one original neoplastic clone. Here, the analysis were extended by loss of heterozygosity (LOH) analysis in the chromosome 9q22.3 region. A total of 85 samples, taken from twenty-two sections of sun-exposed sites, corresponding to normal epidermis, morphological normal cells with positive immuno-staining for the p53 protein (p53 patches), dysplasias, cancer in situ (CIS) and squamous cell carcinomas (SCC) of the skin were analysed. Overall, about 70% of p53 patches had mutations in the p53 gene but not LOH in the p53 gene or 9q22.3 region. Approximately 70% of the dysplasias showed p53 mutations of which about 40% had LOH in the p53 region but not in the 9q22.3 region. In contrast, about 65% of SCC and CIS displayed LOH in the 9q22.3 region, as well as frequent (80%) mutations and/or LOH in the p53 gene. These findings strongly suggest that alterations in the p53 gene is an early event in the progression towards SCC, whereas malignant development involves LOH and alterations in at least one (or several) tumor suppressor genes located in chromosome 9q22.3.
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| 5. |
- Ahmadian, Afshin, et al.
(författare)
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Genotyping by apyrase-mediated allele-specific extension
- 2001
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 29:24
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Tidskriftsartikel (refereegranskat)abstract
- This report describes a single-step extension approach suitable for high-throughput single-nucleotide polymorphism typing applications. The method relies on extension of paired allele-specific primers and we demonstrate that the reaction kinetics were slower for mismatched configurations compared with matched configurations. In our approach we employ apyrase, a nucleotide degrading enzyme, to allow accurate discrimination between matched and mismatched primer-template configurations. This apyrase-mediated allele-specific extension (AMASE) protocol allows incorporation of nucleotides when the reaction kinetics are fast (matched 3'-end primer) but degrades the nucleotides before extension when the reaction kinetics are slow (mismatched 3'-end primer). Thus, AMASE circumvents the major limitation of previous allele-specific extension assays in which slow reaction kinetics will still give rise to extension products from mismatched 3'-end primers, hindering proper discrimination. It thus represents a significant improvement of the allele-extension method. AMASE was evaluated by a bioluminometric assay in which successful incorporation of unmodified nucleotides is monitored in real-time using an enzymatic cascade.
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| 6. |
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| 7. |
- Ahmadian, Afshin, et al.
(författare)
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Method for amplification
- 2005
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Patent (populärvet., debatt m.m.)abstract
- The invention refers to a method for multiplex amplification of at least one specific nucleic acid locus, comprising the steps of: providing at least one oligonucleotide probe pair that is designed so that the first and second probe of the pair anneal to a specific nucleic acid locus on a target molecule, in which pair the first probe has an extendable 3'-end, and a second probe has a 5'-end that is directly or indirectly labelled with a phosphate group; providing a target molecule comprising at least one specific nucleic acid locus; allowing the probe pair to anneal to the target molecule; allowing the 3'-end of the first probe to extend by influence of polymerase by adding a set of three different dNTPs; ligating the 3'-end of the extended first probe to the 5'-end of the second probe. Hereby, a method is provided which allows a high specificity for simultaneous amplification of several loci. Further, the invention involves a kit for use in the method of the invention.
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| 8. |
- Ahmadian, Afshin, et al.
(författare)
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Pyrosequencing : History, biochemistry and future
- 2006
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Ingår i: Clinica Chimica Acta. - : Elsevier BV. - 0009-8981 .- 1873-3492. ; 363:02-jan, s. 83-94
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Forskningsöversikt (refereegranskat)abstract
- Background: Pyrosequencing is a DNA sequencing technology based on the sequencing-by-synthesis principle. Methods: The technique is built on a 4-enzyme real-time monitoring of DNA synthesis by bioluminescence using a cascade that upon nucleotide incorporation ends in a detectable light signal (bioluminescence). The detection system is based on the pyrophosphate released when a nucleotide is introduced in the DNA-strand. Thereby, the signal can be quantitatively connected to the number of bases added. Currently, the technique is limited to analysis of short DNA sequences exemplified by single-nucleotide polymorphism analysis and genotyping. 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. In order to expand the field for pyrosequencing, the read length needs to be improved. Conclusions: Th pyrosequencing system is based on an enzymatic system. There are different current and future applications of this technique.
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| 9. |
- 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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| 10. |
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