A Rapid Method for Analysis of cDNA Synthesis Using Ion-Pair Reversed-Phase High Performance Liquid Chromatography

Journal of Cell and Molecular Research

مقاله 2، دوره 11، شماره 2 - شماره پیاپی 22، خرداد 2020، صفحه 55-58   

نوع مقاله: Research Articles

شناسه دیجیتال (DOI): 10.22067/jcmr.v11i2.83212

نویسندگان

Maryam M. Matin^{* 1}؛ David P. Hornby²

¹Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran

²Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom

چکیده

     We have developed a rapid, quantitative method for analysing the outcome of the first strand synthesis step in cDNA library preparation, yield and molecular weight range of the final cDNA products are determined after size fractionation. This method involves conventional cDNA library construction including all enzymatic steps usually required, but replaces radioactive labelling of nucleic acids with fluorescence detection. The separation and quantification steps all involve ion-pair reversed-phase high performance liquid chromatography (IP RP HPLC). This quantitative method replaces the use of autoradiography and size exclusion chromatography with combined ion-pair reversed-phase high performance liquid chromatography and in line fluorescence detection. The result of this approach is combination of speed with the generation of reproducible, high quality cDNA libraries.

کلیدواژه‌ها

Molecular Biology؛ cDNA؛ library construction؛ IP RP HPLC؛ size fractionation

مراجع

Bashirdes, S. and Lovett, M. (2001) cDNA detection and analysis. Current Opinion in Chemical Biology 5: 15–20.

Breuzovska, K., Dimitrovska, A., Kitanovski, Z., Petrusevska, J., Ribarska, J.T. and Jolevska, S.T. (2010) Development of an ion-pair reversed-phase HPLC method with indirect UV detection for determination of phosphates and phosphites as impurities in sodium risedronate. Journal of AOAC International 93: 1113-1120.

Delmotte, N., Lasaosa, M., Tholey, A., Heinzle, E. and Huber, C.G. (2007) Two-dimensional reversed-phase x ion-pair reversed-phase HPLC: an alternative approach to high-resolution peptide separation for shotgun proteome analysis. Journal of Proteome Research 6: 4363-4373.

Doris, P.A., Oefner, P.J., Chilton, B.S. andHayward-Lester, A. (1998) Quantitative analysis of gene expression by ion-pair high-performance liquid chromatography. Journal of Chromatography A 806: 47-60.

Gubler, U. and Hoffman, B.J. (1983) A simple and very efficient method for generating cDNA libraries. Gene 25: 263-269.

Harbers, M. (2008) The current status of cDNA cloning. Genomics 91: 232-242.

Hillmann, A., Dunne, E. and Kenny, D. (2009) cDNA amplification by SMART-PCR and suppression subtractive hybridization (SSH)-PCR. Methods in Molecular Biology 496: 223-43.

Kiefer, P., Delmotte, N. and Vorholt, J.A. (2011) Nanoscale ion-pair reversed-phase HPLC-MS for sensitive metabolome analysis. Analytical Chemistry 83(3):850-5

Kimmel, A.R. and Berger, S.L. (1987) Preparation of cDNA and the generation of cDNA libraries: overview. Methods in Enzymology 152: 307-316.

Kyriakidesa, D. and Panderi, I. (2007) Development and validation of a reversed-phase ion-pair high-performance liquid chromatographic method for the determination of risedronate in pharmaceutical preparations. Analytica Chimica Acta 584: 153-159.

Matin, M.M. and Hornby, D.P. (2000) A positive selection vector combining tetracycline resistance that eliminates the need for bacterial plating. Analytical Biochemistry 278: 46-51.

Nakajima, K., Kitazume, S., Angata, T., Fujinawa, R., Ohtsubo, K., Miyoshi, E. and Taniguchi, N. (2010) Simultaneous determination of nucleotide sugars with ion-pair reversed-phase HPLC. Glycobiology 20: 865-871.

Okayama, H. and Berg. P. (1982) High-efficiency cloning of full-length cDNA. Molecular and Cellular Biology 2: 161-170.

Peng, S.X. and Dansereau, S.M. (2001) Ion-exchange liquid chromatographic analysis of bisphosphonates by on-line post-column photochemical reaction and spectrophotometric detection. Journal of Chromatography A 914: 105-110.

Roeder, T. (1998) Solid-phase cDNA library construction, a versatile approach. Nucleic Acids Research 26: 3451-3452.

Sambrook, J. and Russell, D. (2001) Molecular Cloning: A laboratory Manual. (3rd ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Wei, X., Dai, G., Liu, Z., Cheng, H., Xie, Z., Marcucci, G., and Chan, K.K. (2006) Metabolism of GTI-2040, a phosphorothioate oligonucleotide antisense, using ion-pair reversed phase high performance liquid chromatography (HPLC) coupled with electrospray ion-trap mass spectrometry. AAPS Journal 8: E743-755.

Ying, S.Y. (2004) Complementary DNA Libraries. Molecular Biotechnology 27: 245-252.

Zhang, L. (2010) Analysis of risedronate and related substances by ion-pair reversed-phase high-performance liquid chromatography with evaporative light-scattering detection. Analytical Sciences 26: 325-329.