Reverse Purification of Genomic DNA

The purification of genomic DNA is a major prerequisite for numerous analytical methods in modern life science and nucleic acid research. Although there are numbers of purification methods, they are often laborious and time-consuming. We report on a newly developed chromatographic separation material for the effective purification of genomic DNA from crude cell extracts.

Method

The separation material consists of special microcapsules with walls of defined porosity. Based on the chromatographic principle of size exclusion, a micro column filled with such microcapsules was designed, which enables a rapid DNA purification in a one step purification procedure.

The excluded fraction, containing only the genomic DNA, is eluted first, whereas the permeable fraction, containing all other components of the cell extract with smaller molecular mass (e.g. proteins, polysaccharides, salts), remains in the column as the elution process stops automatically. Connecting a special concentrator tube to the outlet tip of the column additionally allows for simultaneous collection and concentration of pure DNA.

By this reverse purification technique the product of interest, genomic DNA, is received first. There are no other steps such as binding, washing, dissolving and precipitation of DNA necessary. Just add the cell extract to the column, draw elution buffer through and separate, collect and concentrate DNA in one step.

Applications

The new separation technology is applicable for isolation of genomic DNA from various biological sources. Genomic DNA was successfully isolated from plants (e.g. Arabidopsis thaliana, Zea mays, Triticum aestivum, Daucus carota, Rosa spec., Juglans regia, Quercus robur), animal and human sources (tail, liver and kidney from mouse and pork, HeLa-cells, human blood) and microorganisms (Saccharomyces cereviseae, Streptomyces spec., Anabena variabilis).

Specialized kits with optimal buffers for cell digestion and DNA elution are available. Isolation can also be carried out without additional steps from tissues which are difficult to prepare, e.g. from plants rich in phenolic substances and polysaccharides, which often affect purification by common techniques. The purified DNA, also from smallest sample amounts (e.g. 1 to 25 mg fresh mass), was successfully verified in downstream applications like PCR, sequencing, methylation analysis.

Benefits

The new method is effective, rapid, easy, and provides high-quality genomic DNA. The DNA can be used directly for PCR and all other downstream applications. The simplicity of the handling strongly facilitates automatisation. A marginal consumption of consumables and no need for organic liquids or other hazardous substances make the new development a green product.