Methodology Optimization of DNA extraction from fresh leaf tissues of Melanoxylon brauna (Fabaceae)

Abstract

ABSTRACT. Melanoxylon brauna (Fabaceae -Caesalpinioideae) is an endemic and valuable hardwood tree species in the Brazilian Atlantic rainforest; it is comparable to African ebony wood. We tested three protocols of DNA extraction based on the citrimonium bromide (CTAB) method and evaluated the quantity, purity and integrity of the DNA. We also determined whether these procedures interfere with PCR amplification in order to develop a protocol for M. brauna. We found that the quality and integrity of DNA were improved with the use of proteinase K in the extraction buffer and by modifications in the centrifugation speed. The lowest concentration of DNA was obtained with Doyle and Doyle's protocol (5.42 ng/μL). Ferreira and Grattapaglia's protocol modified for M. brauna provided the most DNA (36.89 ng/μL) and the highest quality DNA (purity ratio of 1.80 nm). The original Ferreira and Grattapaglia protocol provided 13.42 ng/μL DNA; however, the purity ratio (1.44 nm) indicates protein contamination. PCR results showed that Ferreira and Grattapaglia's protocol modified for M. brauna gave satisfactory quantity and purity of DNA for molecular studies.

DNA Extraction and Amplification from Contemporary Polynesian Bark-Cloth

“…The ratio of 260/280 values corresponded to pure DNA samples, except in the case of Tongan bark-cloth, which presented a minimum ratio of 1.39 and Easter Island 1 bark-cloth, which presented a maximum ratio of 2.55. Values below 1.8 can be explained by the presence of contaminants [25], whereas a ratio higher than 2.0 nm indicates that the samples could be contaminated with residual chloroform from the extraction procedure [26]. It is difficult to ascribe ratios over 2.0 to the presence of RNA, as has been previously described [25], since is not very plausible that RNA is still present, especially as single stranded nucleic acid molecules, considering that the bark is subjected to a prolonged and complex treatment during the making of bark-cloth.…”

Short Communication Comparison of methods to isolate DNA from Caesalpinia ferrea

“…PVP has antioxidant properties and is responsible for deproteinization and the removal of polyphenols, tannins, and quinone that are present in large quantities in plants belonging to the Caesalpinioideae family. Borges et al (2012) compared the DNA extracted from another species in the Caesalpinioideae family, Melanoxylon brauna, using the Doyle and Doyle (1987) and Ferreira and Grattapaglia (1998) protocols, and they determined that the DNA extracted using the second protocol was of a better quality and greater quantity. Even though the Clarke et al (1989) protocol utilized higher concentrations of PVP and EDTA in the buffer solution and had the highest concentration of NaCl, which contributes positive ions that neutralize the negative charge of DNA and reduces DNA contamination with polysaccharides, this extraction protocol did not have satisfactory results.…”

A simplified genomic DNA extraction protocol for pre-germination genotyping in rice

“…Moreover, these methods are generally phenol-chloroform-dependent, and may involve the use of hazardous reagents that require special handling and waste disposal (Sambrook et al, 2002). In addition, other protocols use expensive reagents such as proteinase K in the DNA isolation process (Borges et al, 2012), making these procedures difficult to apply on a large scale.…”