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Keçi Sütü Somatik Hücrelerinden Genomik DNA İzolasyonunda Fenol-Kloroform ve Chelex® 100 Ekstraksiyon Yöntemlerinin Karşılaştırılması

Year 2005, Volume: 11 Issue: 01, 16 - 20, 01.01.2005
https://doi.org/10.1501/Tarimbil_0000000491

Abstract

Bu çalışmada, Ankara Üniversitesi Ziraat Fakültesi Zootekni Bölüm İşletmesinde yetiştirilen Saanen–Kilis melezi Akkeçi’lerden ve Konya–Ermenek yöresi özel işletmelerdeki Kıl keçi’lerden temin edilen toplam 30 adet süt örneğinden genomik DNA izolasyonu yapılmıştır. Genomik DNA izolasyonu, fenol–kloroform ile Chelex® 100 ekstraksiyon yöntemleri kullanılarak yapılmıştır. Her iki yöntemle elde edilen genomik DNA’nın konsantrasyonu ve saflık derecesi, 260 ve 280 nm UV ışığı altında absorbans değerlerinden hesaplanmış ve karşılaştırılmıştır. Fenol–kloroform ekstraksiyon yöntemi ile elde edilen DNA konsantrasyonu ortalama, 2406±322 µg/mL ve saflık derecesi A260/A280oranında ortalama, 1.816±0.038 olarak bulunmuştur. Bu durum fenol-kloroform ekstraksiyon yöntemiyle genomik DNA’nın saf olarak elde edildiğini göstermektedir. Chelex® 100 ekstraksiyon yönteminde ise DNA konsantrasyonu ortalama, 590.0±54.9 µg/mL ve elde edilen genomik DNA’nın saflık derecesi ortalama, 1.311±0.012 olarak bulunmuştur. Bu sonuç Chelex® 100 ekstraksiyonu ile elde edilen DNA örneklerinde protein kontaminasyonu olduğunu göstermekte ve DNA’ların çok saf elde edilemediğini ifade etmektedir. Fenol–kloroform ekstraksiyon yöntemi ile elde edilen genomik DNA, % 1.0 ’lik agaroz jelinde tek bant olarak görülmüştür. Ancak Chelex® 100 ekstraksiyon yöntemi ile elde edilen DNA miktarı az olduğu için agaroz jelinde görüntü alınamamıştır

References

  • Amills, M., O. Francino, M. Jansa and A. Sanchez, 1997. Isolation of genomic DNA from milk samples by using Chelex® resin. Journal of Dairy Research, 64: 231-238.
  • Brown, T. A. 1991. Essential Molecular Biology Vol: 1 A Practical Approach. Oxford University Press, p.47-68. New York.
  • Cerit, H., H. Demir, 1999. Sığırlarda kan hücrelerinden DNA ekstraksiyonu için Chelex® 100’ün kullanım olanakları. İstanbul Üniv. Vet. Fak. Derg. 25 (2): 239–244.
  • Chrispeels, M. J. 1973. Molecular techniques and approaches in developmental biology. California University Press, p.128- 150. USA.
  • Ciulla, T. A., R. M. Sklar and S. L. Hauser, 1988. A simple method for DNA purification from peripheral blood. Analytical Biochemistry, 174: 485-488.
  • De, S., R. K. Singh, P. K. Gupta, S. Palia and G. Butchaiah, 2000. Genotyping of dairy animals using DNA from milk somatic cells. Indian Journal of Animal Sciences, 70 (9): 944-946.
  • Goossens, M., Y. Kan, 1981. DNA Analysis in the diagnosis of hemoglobin disorders. Methods in Enzmology, Vol: 76.
  • Jeanpierre, M. 1987. A rapid method for the purification of DNA from blood. Nucleic Acids Research, 15 (22): 9611.
  • Johns, M. B., J. E. Paulus-Thomas, 1989. Purification of human genomic DNA from whole blood using sodium perchlorate in place of phenol. Analytical Biochemistry, 180: 276-278.
  • Lipkin, E., A. Shalom, H. Khatib, M. Soller and A. Friedmann, 1993. Milk as a source of deoxyribonucleic acid and as a substrate for the polymerase chain reaction. Journal of Dairy Sci., 76: 2025-2032.
  • Kehrli, M. E., D. E. Shuster, 1994. Factors affecting milk somatic cells and their role in health of the bovine mammary gland. Journal of Dairy Science, 77: 619-627.
  • Miller, S. A., D. D. Dykes and H. F. Polesky, 1988. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Research, 16 (3): 1215.
  • Montgomery, G. W., J. A. Sise, 1990. Extraction of DNA from sheep white blood cells. New Zealand Journal of Agricultural Research, 33: 437-441.
  • Mullenbach, R., P. J. L Lagoda and C. Welter, 1989. An efficient salt–chloroform extraction of DNA from blood and tissues. Trends in Genetics, 5 (12): 391.
  • Sambrook, J., E. F. Fritsch and T. Maniatis, 1989. Molecular Cloning. A Laboratory Manual Appendixes 2nd edition. Cold Spring Harbor Laboratory Press, p.6.4-6.20 USA.
  • Silhavy, T. J., M. L. Berman and L. W. Enquist, 1984. Experiments with Gene Fusions. Cold Spring Harbor Laboratory Press, 301, p.177-183. USA.
  • Singer-Sam, J., R. L. Tanguay and A. D. Riggs, 1989. Use of chelex to improve the PCR signal from a small number of cell. Amplifications A Forum for PCR Users, 3: 11.
  • Walsh, P. S., D. A. Metzger and R. Higuchi, 1991. Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. BioTechniques, 10 (4): 506- 513.
  • Yükseloğlu, E. H. 1996. HLA – DQA1 Lokusunun Polimeraz Zincir Tepkimesine (PCR) Dayanan 2 Farklı Teknik ile Tiplenmesinin Adli Bilimler Açısından Değerlendirilmesi. Yüksek Lisans Tezi (Yayınlanmamış), İstanbul Üniv. Adli Tıp Enstitüsü.

The Comparison of Phenol-Chloroform and Chelex® 100 Extraction Methods for the Isolation of Genomic DNA From Goat Milk Somatic Cells

Year 2005, Volume: 11 Issue: 01, 16 - 20, 01.01.2005
https://doi.org/10.1501/Tarimbil_0000000491

Abstract

In this study, the isolation of genomic DNA was carried out by using a total of 30 Akkeçi and native hair goat milk samples that were collected from Konya-Ermenek region and University of Ankara, Faculty of Agriculture, Department of Animal Science. The isolation of genomic DNA was performed by using phenol–chloroform and Chelex® 100 extraction methods. The concentration and the purity of genomic DNA obtained with both methods, were determined by spectrophotometric absorption of UV light at 260 nm and 280 nm wave length and the results were compared. The mean DNA concentration obtained from phenol–chloroform extraction method was 2406±322 µg/mL and the mean purity of genomic DNA A260/A280 ratio was 1.816±0.038. These results indicate good deproteinisation in phenol–chloroform extraction method. The mean DNA concentration obtained from Chelex® 100 extraction method was 590.0±54.9 µg/mL and the mean purity of genomic DNA was 1.311±0.012. These results showed that there were some protein contaminations in DNA samples by using Chelex® 100 extraction method. The genomic DNA obtained from phenol–chloroform extraction method, appeared to be a single band on 1 % agarose gel but due to the less amount of genomic DNA obtained in Chelex® 100 extraction method, there was no signal on agarose gel

References

  • Amills, M., O. Francino, M. Jansa and A. Sanchez, 1997. Isolation of genomic DNA from milk samples by using Chelex® resin. Journal of Dairy Research, 64: 231-238.
  • Brown, T. A. 1991. Essential Molecular Biology Vol: 1 A Practical Approach. Oxford University Press, p.47-68. New York.
  • Cerit, H., H. Demir, 1999. Sığırlarda kan hücrelerinden DNA ekstraksiyonu için Chelex® 100’ün kullanım olanakları. İstanbul Üniv. Vet. Fak. Derg. 25 (2): 239–244.
  • Chrispeels, M. J. 1973. Molecular techniques and approaches in developmental biology. California University Press, p.128- 150. USA.
  • Ciulla, T. A., R. M. Sklar and S. L. Hauser, 1988. A simple method for DNA purification from peripheral blood. Analytical Biochemistry, 174: 485-488.
  • De, S., R. K. Singh, P. K. Gupta, S. Palia and G. Butchaiah, 2000. Genotyping of dairy animals using DNA from milk somatic cells. Indian Journal of Animal Sciences, 70 (9): 944-946.
  • Goossens, M., Y. Kan, 1981. DNA Analysis in the diagnosis of hemoglobin disorders. Methods in Enzmology, Vol: 76.
  • Jeanpierre, M. 1987. A rapid method for the purification of DNA from blood. Nucleic Acids Research, 15 (22): 9611.
  • Johns, M. B., J. E. Paulus-Thomas, 1989. Purification of human genomic DNA from whole blood using sodium perchlorate in place of phenol. Analytical Biochemistry, 180: 276-278.
  • Lipkin, E., A. Shalom, H. Khatib, M. Soller and A. Friedmann, 1993. Milk as a source of deoxyribonucleic acid and as a substrate for the polymerase chain reaction. Journal of Dairy Sci., 76: 2025-2032.
  • Kehrli, M. E., D. E. Shuster, 1994. Factors affecting milk somatic cells and their role in health of the bovine mammary gland. Journal of Dairy Science, 77: 619-627.
  • Miller, S. A., D. D. Dykes and H. F. Polesky, 1988. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Research, 16 (3): 1215.
  • Montgomery, G. W., J. A. Sise, 1990. Extraction of DNA from sheep white blood cells. New Zealand Journal of Agricultural Research, 33: 437-441.
  • Mullenbach, R., P. J. L Lagoda and C. Welter, 1989. An efficient salt–chloroform extraction of DNA from blood and tissues. Trends in Genetics, 5 (12): 391.
  • Sambrook, J., E. F. Fritsch and T. Maniatis, 1989. Molecular Cloning. A Laboratory Manual Appendixes 2nd edition. Cold Spring Harbor Laboratory Press, p.6.4-6.20 USA.
  • Silhavy, T. J., M. L. Berman and L. W. Enquist, 1984. Experiments with Gene Fusions. Cold Spring Harbor Laboratory Press, 301, p.177-183. USA.
  • Singer-Sam, J., R. L. Tanguay and A. D. Riggs, 1989. Use of chelex to improve the PCR signal from a small number of cell. Amplifications A Forum for PCR Users, 3: 11.
  • Walsh, P. S., D. A. Metzger and R. Higuchi, 1991. Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. BioTechniques, 10 (4): 506- 513.
  • Yükseloğlu, E. H. 1996. HLA – DQA1 Lokusunun Polimeraz Zincir Tepkimesine (PCR) Dayanan 2 Farklı Teknik ile Tiplenmesinin Adli Bilimler Açısından Değerlendirilmesi. Yüksek Lisans Tezi (Yayınlanmamış), İstanbul Üniv. Adli Tıp Enstitüsü.
There are 19 citations in total.

Details

Primary Language Turkish
Journal Section Research Article
Authors

Fulya Özdil This is me

Ensar Başpınar This is me

Publication Date January 1, 2005
Submission Date January 1, 2005
Published in Issue Year 2005 Volume: 11 Issue: 01

Cite

APA Özdil, F., & Başpınar, E. (2005). Keçi Sütü Somatik Hücrelerinden Genomik DNA İzolasyonunda Fenol-Kloroform ve Chelex® 100 Ekstraksiyon Yöntemlerinin Karşılaştırılması. Journal of Agricultural Sciences, 11(01), 16-20. https://doi.org/10.1501/Tarimbil_0000000491

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