Extraction of DNA suitable for PCR applications from mature leaves of Mangifera indica L.

Abstract

Good quality deoxyribonucleic acid (DNA) is the pre-requisite for its downstream applications. The presence of high concentrations of polysaccharides, polyphenols, proteins, and other secondary metabolites in mango leaves poses problem in getting good quality DNA fit for polymerase chain reaction (PCR) applications. The problem is exacerbated when DNA is extracted from mature mango leaves. A reliable and modified protocol based on the cetyltrimethylammonium bromide (CTAB) method for DNA extraction from mature mango leaves is described here. High concentrations of inert salt were used to remove polysaccharides; Polyvinylpyrrolidone (PVP) and β-mercaptoethanol were employed to manage phenolic compounds. Extended chloroform-isoamyl alcohol treatment followed by RNase treatment yielded 950-1050 µg of good quality DNA, free of protein and RNA. The problems of DNA degradation, contamination, and low yield due to irreversible binding of phenolic compounds and coprecipitation of polysaccharides with DNA were avoided by this method. The DNA isolated by the modified method showed good PCR amplification using simple sequence repeat (SSR) primers. This modified protocol can also be used to extract DNA from other woody plants having similar problems.

AN EFFECTIVE METHOD FOR DNA EXTRACTION OF MATURE LEAF OF SAPODILLA (Manilkara zapota (L.) van Royen)

“…The function of mercaptoethanol is to remove polyphenols, in line with PVP which serves as to bind phenolic components and to suppress oxidation (Utami et al, 2012). CIAA (Chloroform isoamilalcohol) repeated up to three times was aimed to optimize the extraction of DNA of contaminants such as mango extraction studies (Azmat et al 2012). According to Syafaruddin and Santoso (2011), chloroform is organic solvent that can dissolve proteins, lipids and other molecules such as polysaccharides.…”

An improved method to extract DNA from mango Mangifera indica

“…In mango, this problem is exacerbated when using fully expanded mature leaves (Azmat et al 2012). In our new protocol, in the first step, 0.4 M glucose was used, which improved DNA quality, avoiding contamination and browning by polyphenolics, since PVP binds phenolic compounds and β-mercaptoethanol was used as an antioxidant.…”

“…The co-precipitation of polysaccharides was avoided by adding higher concentrations of selective precipitants of nucleic acid, CTAB (0.04 g/mL) and NaCl (3 M) (Dellaporta et al 1983); the latter was used at 1.4 M in our new method. Long-tail surfactants such as CTAB produce a conformational change in the DNA from a "random coil" to a "compact globule" making DNA precipitation more effective (Azmat et al 2012). Phenolic compounds are powerful oxidizing agents and bind covalently to the extracted DNA, making it useless for most molecular manipulations (Porebski et al 1997;Padmalatha & Prasad 2006).…”

“…In our protocol, superfluous cellular proteins were removed by a triple extraction with chloroform-isoamyl alcohol, which also helped to remove different colouring substances, such as chlorophyll, pigments and dyes (Azmat et al 2012). He et al (2007) used RNaseA in 0.1X TE during DNA extraction from mango leaves.…”

Identification and characterization of plasma membrane aquaporins isolated from fiber cells of Calotropis procera

“…Young leaves from putative transgenic plants and control tobacco plants (negative controls) were selected for DNA isolation by the modified cetyl trimethyl ammonium bromide (CTAB) method (Azmat et al, 2012). The isolated DNA was resuspended in 50 µl of ultrapure sterile H 2 O and stored at −20 °C.…”