Abstract
Termostabil bir enzim olan Tgo DNA polimeraz
hipertermofilik bir arkea olan Thermococcus
gorgonarius tarafından sentezlenmektedir. Çalışmamızda 3’-5’ ekzonükleaz
aktivitesiyle yüksek kalitede DNA sentezi gerçekleştiren Tgo DNA polimeraz,
rekombinant olarak üretimi hedeflenmiştir. Bu amaçla, ürettirilen yapay gen
uygun restriksiyon enzimleri ile kesilip aynı enzimlerle kesilmiş olan pET28a
vektör sistemine ligasyon ile birleştirilmiştir. E. coli BL21 hücrelerinde eksprese edilen enzim aktivitesi, PCR
reaksiyonu gerçekleştirilerek kontrol edilmiştir.
References
- Banach-Orlowska M, Fijalkowska IJ, Schaaper RM, Jonczyk P (2005). DNA polymerase II as a fidelity factor in chromosomal DNA synthesis in Escherichia coli. Mol Microbiol 58(1): 61-70.
- Fijalkowska IJ, Schaaper RM, Jonczyk P (2012). DNA replication fidelity in Escherichia coli: a multi-DNA polymerase affair. FEMS Microbiol Rev 36(6): 1105-1121.
- Gawel D, Pham PT, Fijalkowska IJ, Jonczyk P, Schaaper RM (2008). Role of accessory DNA polymerases in DNA replication in Escherichia coli: analysis of the dnaX36 mutator mutant. J Bacteriol 190(5): 1730-42.
- Grogan DW (2015). Understanding DNA repair in hyperthermophilic archaea: persistent gaps and other reasons to focus on the fork. Archaea 2015: 942605.
- Hopfner KP, Eichinger A, Engh RA, Laue F, Ankenbauer W, Huber R, Angerer B (1999). Crystal structure of a thermostable type B DNA polymerase from Thermococcus gorgonarius. Proc Natl Acad Sci Biochemistry 96(7): 3600–3605.
- Ishino S and Ishino Y (2014). DNA polymerases as useful reagents for biotechnology – the history of developmental research in the field. Front Microbiol 5: 465.
- Jozwiakowski SK, Keith BJ, Gilroy L, Doherty AJ, Connolly BA (2014). An archaeal family-B DNA polymerase variant able to replicate past DNA damage: occurrence of replicative and translesion synthesis polymerases within the B family. Nucl Acids Res 42(15): 9949–9963.
- Kovalskaya N, Owensa R, Bakera CJ, Deahl K, Hammond RW (2014) Application of a modified EDTA-mediated exudation technique and guttation fluid analysis for potato spindle tuber viroid RNA detection in tomato plants (Solanum lycopersicum). J Virol Methods 198: 75–81.
- Mitchell KJ, Wood JR, Llamas B, Patricia A, McLenachan PA, Kardailsky O, Scofield RP, Worthy TH, Cooper A (2016). Ancient mitochondrial genomes clarify the evolutionary history of New Zealand’s enigmatic acanthisittid wrens. Mol Phylogenetics Evol 102: 295–304.
- Sambrook J and Russell DW (2001). Molecular cloning. A laboratory manual, Cold Spring Harbor Laboratory Press.
- Yang YL and Polisky B (1993). Suppression of ColEl high-copy-number mutants by mutations in the polA gene of Escherichia coli. J Bacteriol 175: 428-437
- Zhang L, Kang M, Xu J, Huang Y (2015) Archaeal DNA polymerases in biotechnology. Appl Microbiol Biotechnol 99: 6585–6597.
Abstract
A thermostable enzyme Tgo DNA polymerase was produced by a hyperthermophilic archaea, Thermococcus gorgonarius. In this study, it was aimed to produce the Tgo DNA polymerase which synthesizes highly qualified DNA by its 3’-5’-exonuclease activity, by recombinant DNA technology. For this purpose, synthesized synthetic DNA was cut with restriction enzymes and ligated into a pET28a vector. The activity of the enzyme expressed in E. coli BL21 was controlled by PCR reaction.
References
- Banach-Orlowska M, Fijalkowska IJ, Schaaper RM, Jonczyk P (2005). DNA polymerase II as a fidelity factor in chromosomal DNA synthesis in Escherichia coli. Mol Microbiol 58(1): 61-70.
- Fijalkowska IJ, Schaaper RM, Jonczyk P (2012). DNA replication fidelity in Escherichia coli: a multi-DNA polymerase affair. FEMS Microbiol Rev 36(6): 1105-1121.
- Gawel D, Pham PT, Fijalkowska IJ, Jonczyk P, Schaaper RM (2008). Role of accessory DNA polymerases in DNA replication in Escherichia coli: analysis of the dnaX36 mutator mutant. J Bacteriol 190(5): 1730-42.
- Grogan DW (2015). Understanding DNA repair in hyperthermophilic archaea: persistent gaps and other reasons to focus on the fork. Archaea 2015: 942605.
- Hopfner KP, Eichinger A, Engh RA, Laue F, Ankenbauer W, Huber R, Angerer B (1999). Crystal structure of a thermostable type B DNA polymerase from Thermococcus gorgonarius. Proc Natl Acad Sci Biochemistry 96(7): 3600–3605.
- Ishino S and Ishino Y (2014). DNA polymerases as useful reagents for biotechnology – the history of developmental research in the field. Front Microbiol 5: 465.
- Jozwiakowski SK, Keith BJ, Gilroy L, Doherty AJ, Connolly BA (2014). An archaeal family-B DNA polymerase variant able to replicate past DNA damage: occurrence of replicative and translesion synthesis polymerases within the B family. Nucl Acids Res 42(15): 9949–9963.
- Kovalskaya N, Owensa R, Bakera CJ, Deahl K, Hammond RW (2014) Application of a modified EDTA-mediated exudation technique and guttation fluid analysis for potato spindle tuber viroid RNA detection in tomato plants (Solanum lycopersicum). J Virol Methods 198: 75–81.
- Mitchell KJ, Wood JR, Llamas B, Patricia A, McLenachan PA, Kardailsky O, Scofield RP, Worthy TH, Cooper A (2016). Ancient mitochondrial genomes clarify the evolutionary history of New Zealand’s enigmatic acanthisittid wrens. Mol Phylogenetics Evol 102: 295–304.
- Sambrook J and Russell DW (2001). Molecular cloning. A laboratory manual, Cold Spring Harbor Laboratory Press.
- Yang YL and Polisky B (1993). Suppression of ColEl high-copy-number mutants by mutations in the polA gene of Escherichia coli. J Bacteriol 175: 428-437
- Zhang L, Kang M, Xu J, Huang Y (2015) Archaeal DNA polymerases in biotechnology. Appl Microbiol Biotechnol 99: 6585–6597.
Details
| Journal Section | Research Articles |
|---|---|
| Authors | |
| Publication Date | April 28, 2017 |
| Submission Date | February 15, 2017 |
| Published in Issue | Year 2017 Volume: 43 Issue: 1 |
Cite
Journal Owner: On behalf of Selçuk University Faculty of Science, Rector Prof. Dr. Metin AKSOY
Selcuk University Journal of Science Faculty accepts articles in Turkish and English with original results in basic sciences and other applied sciences. The journal may also include compilations containing current innovations.
It was first published in 1981 as "S.Ü. Fen-Edebiyat Fakültesi Dergisi" and was published under this name until 1984 (Number 1-4).
In 1984, its name was changed to "S.Ü. Fen-Edeb. Fak. Fen Dergisi" and it was published under this name as of the 5th issue.
When the Faculty of Letters and Sciences was separated into the Faculty of Science and the Faculty of Letters with the decision of the Council of Ministers numbered 2008/4344 published in the Official Gazette dated 3 December 2008 and numbered 27073, it has been published as "Selcuk University Journal of Science Faculty" since 2009.
It has been scanned in DergiPark since 2016.
Selcuk University Journal of Science Faculty is licensed under a Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) License.