Plant genomic DNA isolation: An art or a science

Abstract

Isolating quality DNA from tissues/cells presents a variety of problems in particular when plants are used as the source material. The specific characteristics of plants like the presence of rigid polysaccharide cell wall, pigments, chemical heterogeneity of secondary metabolites found in diverse species of plants, etc., necessitate special consideration and skill during isolation procedure. Until now, numerous protocols have been published for the purpose, but none is found to be universally applicable. Various factors starting from the selection of source material to the concentration of metabolites present in the plant decide the course of the isolation procedure. The present review is an update of various methods used for plant genomic DNA isolation, and it epitomizes the various problems faced and the solutions made to contend with them during DNA isolation from plant cells.

Comparison between modified DNA extraction protocols and commercial isolation kits in grapevine (Vitis vinifera L.)

“…The ratio of absorbance at 260/280 nm is 1.8 for a pure DNA sample, and a decrease in that ratio indicates contamination by proteins, whereas the presence of RNA increases the ratio (Varma et al, 2007). Generally a ratio between 1.8 and 2.0 indicates uncontaminated DNA (Sambrook and Russell, 2001;Alaey et al, 2005).…”

“…Because of this characteristic, as well as specific pigments and different secondary metabolites, DNA extraction from plant tissues requires particular care and skill during isolation (Varma et al, 2007). For this reason, various DNA extraction protocols have been developed in numerous biotechnological research laboratories using plant tissues (Murray and Thompson, 1980;Doyle and Doyle, 1987;Thomas et al, 1993;Lodhi et al, 1994;Lefort et al, 1998).…”

Isolation of High-Quality DNA from a Desert Plant Reaumuria soongorica

“…Endonucleases released from the vacuoles during the cell lysis process, which are co-isolated with highly viscous polysaccharide, lead to the degradation of DNA and remarkably reduce the yield of extracted DNA (Khanuja et al, 1999. Polyphenols released from the vacuoles during the cell lysis process are oxidized by cellular oxidases and undergo irreversible interactions with nucleic acids causing browning of the DNA (Varma et al, 2007;Moyo et al, 2008;Khanuja et al, 1999;Porebski et al, 1997). The presence of gelling polysaccharides prevents complete dissolution of nucleic acids and imparts a viscous constituency to the DNA making it stick to the wells during gel electrophoresis (Barnell et al, 1998;Diadema et al, 2003;Varma et al, 2007).…”

METODE EKSTRAKSI DNA CABAI (<em>Capsicum annuum</em> L.) MENGGUNAKAN MODIFIKASI BUFFER CTAB (CETHYL TRIMETHYL AMMONIUM BROMIDE) TANPA NITROGEN CAIR

“…Sementara itu senyawa β-merkaptoetanol selain berperan sebagai antioksidan juga berperan dalam mendegradasi protein (Varma et al 2007). Senyawa tersebut akan membantu mengurai ikatan sulfat pada struktur protein sehingga protein dapat dieliminasi dengan mudah (Mornkham et al 2012).…”

“…Selanjutnya DNA yang berada di fase air (supernatan) dan telah terbebas dari protein lalu dipindahkan pada tabung baru dan diberi garam natrium asetat. Menurut Varma et al (2007) penambahan garam dengan konsentrasi tinggi akan meningkatan kelarutan senyawa polisakarida pada alkohol sehingga polisakarida tidak akan mengendap bersama DNA saat disentrifugasi. Selain natrium asetat, garam lain yang biasa digunakan pada proses ekstraksi DNA adalah NaCl (Fang et al 1992;Lodhi et al 1994) dan ammonium asetat (Mӧller et al 1992;Singh & Kumar 2012 Hasil uji kuantitatif dengan alat Nanodrop Spektrofotometer menunjukkan bahwa DNA cabai yang diekstraksi pada penelitian ini memiliki kisaran konsentrasi dari 227 ng/µlpada sampel F2 No.…”