Document Type : Research paper

Authors

1 Department of Agronomy and Plant Breeding Sciences, College of Aburaihan, University of Tehran

2 Department of Agronomy and Plant Breeding Sciences, University of Kurdistan- Faculty of Agriculture, Sanandaj, Iran

10.22059/ijhst.2021.329767.500

Abstract

In most procedures that involve gene cloning, after the amplification of a target gene by PCR or by Real-time PCR, the purification of the trapped
gene on agarose gel is a crucial stage. There are various methods for extracting genes from agarose gel by removing other contaminants. We isolated the amplified PqHMGR gene (derived from Ginseng (Panax quinquefolius)) from agarose gel by a quasi-electrophoresis device (similar to electro-elution technique). Moreover, the efficiency of this new approach was compared with that of the commercial kit ‘Silica Bead DNA Gel Extraction’ (Thermo Scientific American Company). Ligation to the PTG-19 plasmid and cloning in E. coli bacteria were also done. The results showed successful isolations of targeted DNA, along with a high efficiency in producing recombinant DNA and in concluding a successful cloning procedure through this new device. The invented method provided a better purification ability than the commercial kit, but because of using the TAE 1X buffer as the purified gene storage solution, the plasmid and bacterial transformation rates were slower than the commercial kit method. It was found that using the new method for the purification of nucleotide sequences by electrophoresis and electrophoresis buffer is feasible, and that these purified fragments can be applied in cloning and sequencing. Using the TAE 1X buffer instead of distilled water did not cause problems in gene binding to PTG-19 plasmid. It also allowed a successful transformation of E. coli bacteria by the modified plasmid. Nonetheless, using TAE 1X buffer reduced the modification rate of the PTG-19 plasmid and decreased the rate of E. coli transformation by the modified plasmid.
Abbreviations:
5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal), Complementary DNA (cDNA), Diethyl Pyrocarbonate (DEPC), Escherichia coli (E. coli), Ethylenediaminetetraacetic acid (EDTA), Isopropyl ß-D-1-thiogalactopyranoside (IPTG), LB (Luria Broth), Optical density 260 (OD260), Optical density 280 (OD280), Panax quinquefolius HMGR (PqHMGR), Polymerase chain reaction (PCR), Reverse transcription polymerase chain reaction (RT-PCR), Tris/Borate/EDTA (TBE), Tris-acetate-EDTA. 1X (TAE).

Keywords

Ausubel M BR, Kingston RE, Moore DD, Seidman JG, Struhl K. (1998) Curr Protoc Mol Biol. Wiley, New York
Bimboim H, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7(6), 1513-1523
Blin N, Gabain A, Bujard H (1975) Isolation of large molecular weight DNA from agarose gels forfurther digestion by restriction enzymes. FEBS Lett 53(1), 84-86
Boom R, Sol C, Salimans M, Jansen C, Wertheim-van Dillen P, Van der Noordaa J (1990) Rapid and simple method for purification of nucleic acids. J. Clin. Microbiol 28(3), 495-503
Bostian KA, Lee RC, Halvorson HO (1979) Preparative
Saed Mocheshi, Izadi Darbandi, Namjoo and Saed Mocheshi Int. J. Hort. Sci. Technol. 2022 9(4): 477-486
486
fractionation of nucleic acids by agarose gel electrophoresis. Anal. Biochem 95(1), 174-182
Chen CW, Thomas Jr CA (1980) Recovery of DNA segments from agarose gels. Anal. Biochem 101(2), 339-341
Chong S, Garcia G (1994) An oligonucleotide-directed, in vitro mutagenesis method using ssDNA and preferential DNA amplification of the mutated strand. Biotechniques 17(4), 719-20, 722, 724-5
Dretzen G, Bellard M, Sassone-Corsi P, Chambon P (1981) A reliable method for the recovery of DNA fragments from agarose and acrylamide gels. Anal. Biochem 112(2), 295-298
Feng Y, Ortega Y, He G, Das P, Xu M, Zhang X, Fayer R, Gatei W, Cama V, Xiao L (2007) Wide geographic distribution of Cryptosporidium bovis and the deer-like genotype in bovines. Vet. Parasitol 144(1-2), 1-9
Girvitz S, Bacchetti S, Rainbow A, Graham F (1980) A rapid and efficient procedure for the purification of DNA from agarose gels. Anal. Biochem 106(2), 492-496
Henrich B, Lubitz W, Fuchs E (1982) Use of benzoylated-naphthoylated DEAE-cellulose to purify and concentrate DNA eluted from agarose gels. J. Biochem. Bioph. Methods 6(2), 149-157
Marko M, Chipperfield R, Birnboim H (1982) A procedure for the large-scale isolation of highly purified plasmid DNA using alkaline extraction and binding to glass powder. Anal. Biochem 121(2), 382-387
Roberts RJ, Murray K (1976) Restriction endonuclease. Crit Rev Biochem 4(2), 123-164
Sambrook H (1989) Molecular cloning: a laboratory manual. Cold Spring Harb, NY.
Sharp PA, Sugden B, Sambrook J (1973) Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose-ethidium bromide electrophoresis. Biochemistry 12(16), 3055-3063
Smith HO (1980) [46] Recovery of DNA from gels. Method Enzmol. vol 65. Elsevier, pp 371-380
Strongin AY, Kozlov YI, Debabov V, Arsatians R, Zlochevsky M (1977) A rellable technique for large-scale DNA separation. Anal. Biochem 79(1-2), 1-10
Sulaiman IM, Hira PR, Zhou L, Al-Ali FM, Al-Shelahi FA, Shweiki HM, Iqbal J, Khalid N, Xiao L (2005) Unique endemicity of cryptosporidiosis in children in Kuwait. J CLIN MICROBIOL 43(6), 2805-2809
Tabak H, Flavell R (1978) A method for the recovery of DNA from agarose gels. Nucleic Acids Res 5(7), 2321-2331
Tautz D, Renz M (1983) An optimized freeze-squeeze method for the recovery of DNA fragments from agarose gels. Anal. Biochem 132(1), 14-19
Thuring R, Sanders J, Borst P (1975) A freeze-squeeze method for recovering long DNA from agarose gels. Anal. Biochem 66(1), 213-220
Vogelstein B, Gillespie D (1979) Preparative and analytical purification of DNA from agarose. PNAS USA 76(2), 615-619
Wienand U, Schwarz Z, Feix G (1979) Electrophoretic elution of nucleic acids from gels adapted for subsequent biological tests: Application for analysis of mRNAs from maize endosperm. FEBS Lett 98(2), 319-323
Wu Q, Song J, Sun Y, Suo F, Li C, Luo H, Liu Y, Li Y, Zhang X, Yao H (2010) Transcript profiles of Panax quinquefolius from flower, leaf and root bring new insights into genes related to ginsenosides biosynthesis and transcriptional regulation. Physiol. Plant 138(2), 134-149
Yang RC-A, Lis J, Wu R (1979) [10] Elution of DNA from agarose gels after electrophoresis. Method Enzmol. vol 68. Elsevier, pp 176-182
Zassenhaus HP, Butow, R.A. and Hannon, Y .P. (1982) Anal Biochem 25, 125-130