Mevlut ARSLAN, Erdem TEZCAN, Haluk CAMCI, Murat Kemal AVCI

Effect of DNA Concentration on Band Intensity and Resolution in Agarose Gel Electrophoresis

Objective: Agarose gel electrophoresis (AGE) is a widely used method for separating, identifying, and purifying nucleic acids. It has been used intensively in molecular researches and biotechnological applications. In AGE, band resolution and band quality are important parameters for researchers. Until now, many factors such as comb thickness, gel concentration, voltage and buffers have been reported to influence resolution and band quality in AGE. However, effect of DNA concentration on band resolution and intensity in AGE is unclear. Therefore, aim of the study was to investigate the effect of DNA concentration on AGE results in detail. Materials and Methods: Different concentrations of DNA Marker and specific DNA fragments obtained by PCR were analyzed, but the loaded total DNA quantity was not changed. Furthermore, the effect of DNA concentration on AGE was also investigated by designed gel combs with different thicknesses and wideness. Findings: It was shown that DNA concentration did not affect gel resolution, and the effect was not changed by comb thickness and wideness. Also, 2-fold dilution did not affect band intensities while 8-fold dilution significantly affected band intensities of all tested DNA fragments. Conclusion: Concentrating samples, at least 8-fold, and using thin and narrow gel combs can be used for desired results in AGE.

Anahtar Kelimeler:

Agarose gel electrophoresis, DNA concentration, band quality, band intensity, gel resolution

Effect of DNA Concentration on Band Intensity and Resolution in Agarose Gel Elec-trophoresis

Objective: Agarose gel electrophoresis (AGE) is a widely used method for separating, identifying, and purifying nucleic acids. It has been used intensively in molecular researches and biotechnological applications. In AGE, band resolution and band quality are important parameters for researchers. Until now, many factors such as comb thickness, gel concentration, voltage and buffers have been reported to influence resolution and band quality in AGE. However, effect of DNA concentration on band resolution and intensity in AGE is unclear. Therefore, aim of the study was to investigate the effect of DNA concentration on AGE results in detail. Material and Method: Different concentrations of DNA Marker and specific DNA fragments obtained by PCR were analyzed, but the loaded total DNA quantity was not changed. Furthermore, the effect of DNA concentra-tion on AGE was also investigated by designed gel combs with different thicknesses and wideness. Results: It was shown that DNA concentration did not affect gel resolution, and the effectwas not changed by comb thickness and wideness. Also, 2-fold dilution did not affect band intensities while 8-fold dilution signifi-cantly affected band intensities of all tested DNA fragments. Conclusion: Concentrating samples, at least 8-fold, and using thin and narrow gel combs can be used for de-sired results in AGE.

Keywords:

Agarose gel electrophoresis, DNA concentration, Band quality, Band intensity, Gel resolution,

PDF

___

Aaij C, Borst P. The gel electrophoresis of DNA. Biochim Biophys Acta 1972; 269:192-200
Arslan M. A new primer designing for PCR-RFLP analysis of A and B genetic variants of bovine kappa-casein. Harran Üniversitesi Veteriner Fakültesi Dergisi 2020; 9:6-11
Bachvaroff R, McMaster PR. Separation of microsomal RNA into five bands during agar electrophoresis. Science 1964; 143:1177-1179
Greaser ML, Warren CM. Protein electrophoresis in agarose gels for separating high molecular weight proteins. Methods Mol Biol 2012; 869:111-118
Green MR, Sambrook J. Analysis of DNA by Agarose Gel Electrophoresis. Cold Spring Harbor Protocols 2019; 2019
Hall AC. A comparison of DNA stains and staining methods for Agarose Gel Electrophoresis. bioRxiv 2020; 568253
Helling RB, Goodman HM, Boyer HW. Analysis of endonuclease R-EcoRI fragments of DNA from lambdoid bacteriophages and other viruses by agarose-gel electrophoresis. J Virol 1974; 14:1235-1244
Huang Q, Baum L, Fu WL. Simple and practical staining of DNA with GelRed in agarose gel electrophoresis. Clinical Laboratory 2010; 56:149-152
Huang Q, Fu WL. Comparative analysis of the DNA staining efficiencies of different fluorescent dyes in preparative agarose gel electrophoresis. Clin Chem Lab Med 2005; 43:841-842
Johnson PH, Grossman LI. Electrophoresis of DNA in agarose gels. Optimizing separations of conformational isomers of double- and single-stranded DNAs. Biochemistry 1977; 16:4217-4225
Lai E, Birren BW, Clark SM, Simon MI, Hood L. Pulsed field gel electrophoresis. Biotechniques 1989; 7:34-42
Lee PY, Costumbrado J, Hsu C-Y, Kim YH. Agarose gel electrophoresis for the separation of DNA fragments. Journal of Visualized Experiments 2012; 3923
Lee SV, Bahaman AR (2012) Discriminatory power of agarose gel electrophoresis in DNA fragments analysis. In: Magdeldin S (ed) Gel Electrophoresis Principles and Basics. IntechOpen, pp 41-55
Li C, Arakawa T. Agarose native gel electrophoresis of proteins. Int J Biol Macromol 2019; 140:668-671
Mathew MK, Smith CL, Cantor CR. High-resolution separation and accurate size determination in pulsed-field gel electrophoresis of DNA. 1. DNA size standards and the effect of agarose and temperature. Biochemistry 1988; 27:9204-9210
Miller SE, Taillon-Miller P, Kwok PY. Cost-effective staining of DNA with SYBR green in preparative agarose gel electrophoresis. Biotechniques 1999; 27:34-36
Moore D, Dowhan D. Purification and Concentration of DNA from Aqueous Solutions. Curr Protoc Mol Biol 2002; 59:2.1.1-2.1.10
Philippsen P, Zachau G. Partial degradation of transfer RNAs and transfer RNA fragments by spleen phosphodiesterase as studied by disc electrophoretic methods. Biochim Biophys Acta 1972; 277:523-538
R Development Core Team (2017) R: A Language and Environment for Statistical Computing In. R Foundation for Statistical Computing, Austria, Vienna
Ross PD. Electrophoresis of DNA. I. On a relationship between electrophoresis and donnan equilibrium experiments on DNA. Biopolymers 1964; 2:9-14
Sambrook J, Russell DW (2001) Molecular Cloning: A Laboratory Manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York, USA
Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nature Methods 2012; 9:671-675
Serwer P. Agarose gels: Properties and use for electrophoresis. Electrophoresis 1983; 4:375-382
Sharp PA, Sugden B, Sambrook J. Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose--ethidium bromide electrophoresis. Biochemistry 1973; 12:3055-3063
Sigmon J, Larcom LL. The effect of ethidium bromide on mobility of DNA fragments in agarose gel electrophoresis. Electrophoresis 1996; 17:1524-1527
Slater GW, Noolandi J. The biased reptation model of DNA gel electrophoresis: mobility vs molecular size and gel concentration. Biopolymers 1989; 28:1781-1791
Smith DR. Agarose gel electrophoresis. Methods Mol Biol 1993; 18:433-438
Stellwagen NC. Electrophoresis of DNA in agarose gels, polyacrylamide gels and in free solution. Electrophoresis 2009; 30 Suppl 1:188-195
Thorne HV. Electrophoretic characterization and fractionation of polyoma virus DNA. J Mol Biol 1967; 24:203-211