摘要
Extraction of DNA from fresh tissues is routine in studies of tropical forest species, but DNA extraction from wood is considered as difficult due to its highly degraded nature and adequate quality of genomic DNA extraction is essential for molecular studies. Very few studies have validated the potential for isolating DNA from dried wood (Heartwood and Sapwood). Wood genomic DNA extraction is difficult from mature timber (Teak (Tectona grandis f;verbanaceae), Black Rosewood (Dalbergia latifolia f;Fabaceae) Ben Teak (Lagerstroemia lanceolata f;Lytheraceae) tissues due to presence of high quantity of secondary metabolites polyphenols, tannins and terpenoids and protein inhibitors. Mostly in laboratories DNA extraction kits are available but by using kits, DNA yield is very low and it is quite expensive too. We have standardized and validated the DNA extraction through manual protocol which is applicable for Bark, Sapwood and Heartwood samples of tree species which contains huge amount of inflexible tissues and fibers. The quality of the DNA was tested by spectrophotometer, gel electrophoresis and PCR (ISSR and SSR) amplification. An avrage DNA yield for heartwood ranges from 0.186 - 0.166 μg/μL and sapwood was ranges from 0.26 - 0.244 μg/μL. Modification of CTAB method was by addition of polyvinylpyrrolidone (PVP) appx 0.25%, variation in Rnase concentration, proteinase treatment with different concentration and incubation time. In order to evaluate the standardized wood genomic DNA extraction protocol, we compared it with the mature leaf and core samples (heartwood and sapwood) of the same timber species. The outcome was also quantified and proved by means of polymerase chain reaction analysis by using ISSR and SSR microsatellite markers conducted with isolated pure DNAs. This modified protocol made increased yield and purity of wood total genomic DNA and facilitate the important application of forensic timber species effort.
Extraction of DNA from fresh tissues is routine in studies of tropical forest species, but DNA extraction from wood is considered as difficult due to its highly degraded nature and adequate quality of genomic DNA extraction is essential for molecular studies. Very few studies have validated the potential for isolating DNA from dried wood (Heartwood and Sapwood). Wood genomic DNA extraction is difficult from mature timber (Teak (Tectona grandis f;verbanaceae), Black Rosewood (Dalbergia latifolia f;Fabaceae) Ben Teak (Lagerstroemia lanceolata f;Lytheraceae) tissues due to presence of high quantity of secondary metabolites polyphenols, tannins and terpenoids and protein inhibitors. Mostly in laboratories DNA extraction kits are available but by using kits, DNA yield is very low and it is quite expensive too. We have standardized and validated the DNA extraction through manual protocol which is applicable for Bark, Sapwood and Heartwood samples of tree species which contains huge amount of inflexible tissues and fibers. The quality of the DNA was tested by spectrophotometer, gel electrophoresis and PCR (ISSR and SSR) amplification. An avrage DNA yield for heartwood ranges from 0.186 - 0.166 μg/μL and sapwood was ranges from 0.26 - 0.244 μg/μL. Modification of CTAB method was by addition of polyvinylpyrrolidone (PVP) appx 0.25%, variation in Rnase concentration, proteinase treatment with different concentration and incubation time. In order to evaluate the standardized wood genomic DNA extraction protocol, we compared it with the mature leaf and core samples (heartwood and sapwood) of the same timber species. The outcome was also quantified and proved by means of polymerase chain reaction analysis by using ISSR and SSR microsatellite markers conducted with isolated pure DNAs. This modified protocol made increased yield and purity of wood total genomic DNA and facilitate the important application of forensic timber species effort.
