Standardization of DNA Extraction Method from Mature Dried Leaves and ISSR-PCR Conditions for <i>Melia dubia</i> Cav. —A Fast Growing Multipurpose Tree Species

Abstract

Melia dubia Cav. of family Meliaceae is a fast growing, high value tree species native to India. Isolating DNA from matured dried leaves of M. dubia was difficult due to accumulation of secondary metabolites, majorly polyphenolics, which resulted in dark brown to black colour of the pellet. In this study, a modified STE-(Sucrose, Tris-HCl and Ethylene Diamine Tetra Acetic Acid) CTAB (hexadecyltrimethylammonium bromide) method was standardized for removal of polyphenolics. The protocol developed yielded 200 -1000 ng/µl of quality DNA without any impurities as evident by A260/280 ratio ranging from 1.75 -2.0. It was also suitable for extracting quality DNA from other members of Meliaceae like Azadirachta indica and Melia azedarach. In downstream applications, the extracted DNA was used for PCR amplification by using ISSR and SSR markers. ISSR PCR conditions were optimized in a reaction volume of 25 µl, consisting of 30 ng of template DNA, 1.5 mM MgCl2, 200 µM of each of dNTPs and 2 U of Taq polymerase. The best amplification was observed and the same was applicable for SSR markers.

An Easy, Quick and Cost Effective Method of High Quality DNA Extraction from Mungbean [Vigna radiata (L.) Wilczek] Without Liquid Nitrogen

“…Precipitated DNA can be hooked out of the solution or collected by centrifugation. In the present study, the extracted DNA was free of phenols as the pellet obtained was clean and white to pale yellow in colour while usually in pulses brown colored DNA pellet is obtained due to presence of phenols and the quality of DNA isolated was comparable to other such recent studies (Laxmi prasanna et al, 2013;Healey et al, 2014;Rawat et al, 2016;Sajib et al, 2017). Moreover, here we used young and tender leaves as they contain less phenol as compared to mature leaves which contain more phenol content and the DNA obtained is generally brown in color.…”

Species delimitation of Melia dubia Cav. from Melia azedarach L. complex based on DNA barcoding

“…All of the collected accessions of Melia were successfully sequenced, even though they were reported to contain diverse metabolites; these can copurify and irreversibly bind with nucleic acids to affect PCR amplification of the barcode markers (Rawat et al 2016). Though the mononucleotide repeats were reported to cause problems in sequencing of trnH-psbA marker from plants (Devey et al 2009), such problems were not encountered in Melia, probably due to smaller size of the repeats and low frequency of occurrence.…”

An Effective Wood DNA Extraction Protocol for Three Economic Important Timber Species of India

“…3 -5 μM thickness was prepared from each samples using paper cutter or sliding microtome to produce small chips of wood samples. Kept the chipped slices for drying 5 -10 min before imperilling to for CTAB based protocols for genomic DNA extraction which comprised of CTAB method [17], DNAsure plant mini kit (Nucleopore) [18], and CTAB method (protocol 3) developed at IWST laboratory. Protocol 1 are executed earlier while the protocol 2 are kit manufacturer based and all are a routine based protocol published in number of papers so here only the modified standardised developed i.e., protocol 3 (modified CTAB) has been described below.…”