A fully automatable enzymatic method for DNA extraction from plant tissues

Division of Chemistry, Lomonosov Moscow State University, Moskva, Moscow, Russia
BMC Plant Biology (Impact Factor: 3.81). 02/2005; 5(1):23. DOI: 10.1186/1471-2229-5-23
Source: PubMed


DNA extraction from plant tissues, unlike DNA isolation from mammalian tissues, remains difficult due to the presence of a rigid cell wall around the plant cells. Currently used methods inevitably require a laborious mechanical grinding step, necessary to disrupt the cell wall for the release of DNA.
Using a cocktail of different carbohydrases, a method was developed that enables a complete digestion of the plant cell walls and subsequent DNA release. Optimized conditions for the digestion reaction minimize DNA shearing and digestion, and maximize DNA release from the plant cell. The method gave good results in 125 of the 156 tested species.
In combination with conventional DNA isolation techniques, the new enzymatic method allows to obtain high-yield, high-molecular weight DNA, which can be used for many applications, including genome characterization by AFLP, RAPD and SSR. Automation of the protocol (from leaf disks to DNA) is possible with existing workstations.

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    • "To investigate potential begomoviral infection, we extracted total DNA from the leaves of 25 infected plants using the cetyl trimethyl ammonium bromide (CTAB) method (Manen et al. 2005). PCR was performed separately for each sample by using a pair of degenerate primers specific to the coat protein region of begomovirus [ "
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    • "During the survey for begomovirus infection 20–30% yellow vein disease was observed on Lantana camara in the nursery garden of Sirsa, Haryana province, India. To investigate the potential begomoviral infection, total DNA was extracted from leaves of 32 infected plants using the cetyl trimethyl ammonium bromide (CTAB) method [4]. To confirm the presence of begomovirus in infected samples PCR was performed using a pair of degenerate primers specific to the coat protein region of begomovirus. "

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