Abstract
Amaç: Belirli mutasyonlara neden olan moleküler değişiklikler tümör gelişimi ve sağkalımı için gereklidir. Bu moleküler hedeflerin doğru analiz edilmesi farklı kanser tiplerinde tanı, erken teşhis, prognoz tahmini ve tedavide yol gösterme açısından önemlidir. Bu nedenle, moleküler belirteçlerin hassas tayini için Sanger dizilemenin yanı sıra mini-dizileme protokolünü de optimize ederek kullanmayı amaçladık.
Yöntem: Gliom örneklerinden elde edilmiş genomik DNAlar kullanılarak IDH1 R132, IDH2 R140/R172 ve TERT promotör C228/C250 mutasyonları için Sanger dizileme ve mini-dizileme yapılmıştır. Mini-dizileme reaksiyonları mutasyon saptama primerleri ile “SnaPshot Multiplex Ready Reaction Mix” kullanılarak gerçekleştirilmiş ve otomatik kapiler elektroforezinde yürütülmüştür. Çoklu eğriler “GeneMapper Software” kullanılarak analiz edilmiştir.
Bulgular: Mini-dizileme analizlerinde, yabanıl tip alellere ve farklı mutasyonlara ait eğriler tespit edilmiştir. Yabanıl tip piklerin yanında yer alan eğriler, o noktada varyasyona işaret etmekte ve mutasyon renge göre tanımlanmaktadır.
Sonuç: Moleküler belirteçlerin kanserde tayini oldukça önemlidir. Minidizileme yöntemi moleküler hedeflerin belirlenmesinde güvenilir bir yöntemdir.
References
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- 5. Waitkus MS, Diplas BH, Yan H. Biological Role and Therapeutic Potential of IDH Mutations in Cancer. Cancer Cell 2018;34(2):186-195.
- 6. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100(1):57-70.
- 7. Vinagre J, Almeida A, Pópulo H, Batista R, Lyra J, Pinto V, et al. Frequency of TERT promoter mutations in human cancers. Nat Commun 2013;4:2185.
- 8. Killela PJ, Reitman ZJ, Jiao Y, Bettegowda C, Agrawal N, Diaz LA, et al. TERT promoter mutations occur frequently in gliomas and a subset of tumors derived from cells with low rates of self-renewal. Proc Natl Acad Sci USA 2013;110(15):6021-6.
- 9. Tsiatis AC, Norris-Kirby A, Rich RG, Hafez MJ, Gocke CD, Eshleman JR, et al. Comparison of Sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS mutations. J Mol Diagn 2010;12(4):425–432.
- 10. Syvänen AC, Aalto-Setälä K, Harju L, Kontula K, Söderlund H. A primer-guided nucleotide incorporation assay in the genotyping of apolipoprotein E. Genomics 1990;8(4):684-92.
- 11. Syvänen AC. From gels to chips: "minisequencing" primer extension for analysis of point mutations and single nucleotide polymorphisms. Hum Mutat 1999;13(1):1-10.
- 12. Pastinen T, Kurg A, Metspalu A, Peltonen L, Syvänen AC. Minisequencing: a specific tool for DNA analysis and diagnostics on oligonucleotide arrays. Genome Res 1997;7(6):606-14.
- 13. Carvalho CMB, Pena SDJ. Optimization of a multiplex minisequencing protocol for population studies and medical genetics. Genet Mol Res 2005;4 (2):115-125.
- 14. Balss J, Meyer J, Mueller W, Korshunov A, Hartmann C, von Deimling A. Analysis of the IDH1 codon 132 mutation in brain tumors. Acta Neuropathol 2008;116(6):597-602.
- 15. Sanger F, Coulson AR. A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. J Mol Biol 1975;94(3):441-8.
- 16. Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 1977;74(12): 5463–5467.
- 17. Fiorentino F, Magli MC, Podini D, Ferraretti AP, Nuccitelli A, Vitale N, et al. The minisequencing method: an alternative strategy for preimplantation genetic diagnosis of single gene disorders. Mol Hum Reprod 2003;9(7):399-410.
Abstract
Purpose: Molecular alterations leading to specific mutations are essential for tumor development and survival. Accurate analysis of these molecular targets is important for diagnosis, early detection, forecasting of prognosis and aiding in treatment of different cancer types. Therefore, for sensitive analysis of molecular markers, we aimed to optimize and use minisequencing protocols besides Sanger sequencing.
Methods and Materials: Sanger sequencing and minisequencing were performed for IDH1 R132, IDH2 R140/R172 and TERT promoter C228/C250 mutations using genomic DNA isolated from glioma samples. Minisequencing reactions were performed with detection primers using SnaPshot Multiplex Ready Reaction Mix and run on automated capillary electrophoresis. Multiplex peaks were analyzed with GeneMapper Software.
Results: In the multiplex minisequencing analyses, peaks corresponding to wild type alleles and different mutations were detected. The presence of the peaks next to the wild type peaks point to the presence of variations in that location and the nature of the mutation can be identified according to the color.
Conclusions: Identification of molecular markers in cancer is very important. Minisequencing is a reliable method for the detection of molecular targets.
References
- 1. Chakravarthi BV, Nepal S, Varambally S. Genomic and Epigenomic Alterations in Cancer. Am J Pathol 2016;186(7):1724-35.
- 2. Nair M, Sandhu SS, Sharma AK. Cancer molecular markers: A guide to cancer detection and management. Semin Cancer Biol 2018;52(Pt 1):39-55.
- 3. Dang L, White DW, Gross S, Bennett BD, Bittinger MA, Driggers EM, et al. Cancer- associated IDH1 mutations produce 2-hydroxyglutarate. Nature 2009;462(7274):739-44.
- 4. Xu W, Yang H, Liu Y, Yang Y, Wang P, Kim SH, et al. Oncometabolite 2-hydroxyglutarate is a competitive inhibitor of alpha-ketoglutarate-dependent dioxygenases. Cancer Cell 2011;19(1):17- 30.
- 5. Waitkus MS, Diplas BH, Yan H. Biological Role and Therapeutic Potential of IDH Mutations in Cancer. Cancer Cell 2018;34(2):186-195.
- 6. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100(1):57-70.
- 7. Vinagre J, Almeida A, Pópulo H, Batista R, Lyra J, Pinto V, et al. Frequency of TERT promoter mutations in human cancers. Nat Commun 2013;4:2185.
- 8. Killela PJ, Reitman ZJ, Jiao Y, Bettegowda C, Agrawal N, Diaz LA, et al. TERT promoter mutations occur frequently in gliomas and a subset of tumors derived from cells with low rates of self-renewal. Proc Natl Acad Sci USA 2013;110(15):6021-6.
- 9. Tsiatis AC, Norris-Kirby A, Rich RG, Hafez MJ, Gocke CD, Eshleman JR, et al. Comparison of Sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS mutations. J Mol Diagn 2010;12(4):425–432.
- 10. Syvänen AC, Aalto-Setälä K, Harju L, Kontula K, Söderlund H. A primer-guided nucleotide incorporation assay in the genotyping of apolipoprotein E. Genomics 1990;8(4):684-92.
- 11. Syvänen AC. From gels to chips: "minisequencing" primer extension for analysis of point mutations and single nucleotide polymorphisms. Hum Mutat 1999;13(1):1-10.
- 12. Pastinen T, Kurg A, Metspalu A, Peltonen L, Syvänen AC. Minisequencing: a specific tool for DNA analysis and diagnostics on oligonucleotide arrays. Genome Res 1997;7(6):606-14.
- 13. Carvalho CMB, Pena SDJ. Optimization of a multiplex minisequencing protocol for population studies and medical genetics. Genet Mol Res 2005;4 (2):115-125.
- 14. Balss J, Meyer J, Mueller W, Korshunov A, Hartmann C, von Deimling A. Analysis of the IDH1 codon 132 mutation in brain tumors. Acta Neuropathol 2008;116(6):597-602.
- 15. Sanger F, Coulson AR. A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. J Mol Biol 1975;94(3):441-8.
- 16. Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 1977;74(12): 5463–5467.
- 17. Fiorentino F, Magli MC, Podini D, Ferraretti AP, Nuccitelli A, Vitale N, et al. The minisequencing method: an alternative strategy for preimplantation genetic diagnosis of single gene disorders. Mol Hum Reprod 2003;9(7):399-410.
Details
| Primary Language | English |
|---|---|
| Subjects | Biochemistry and Cell Biology (Other) |
| Journal Section | Research Articles |
| Authors | |
| Publication Date | July 1, 2021 |
| Submission Date | September 7, 2020 |
| Published in Issue | Year 2021Volume: 12 Issue: 3 |
