DNA ISOLATION AND PURIFICATION METHODS

Cesium chloride density gradients

Genomic DNA can be purified by centrifugation through a cesium chloride (CsCl) density gradient. Cells are lysed using a detergent, and the lysate is alcohol precipitated. Resuspended DNA is mixed with CsCl and ethidium bromide and centrifuged for several hours. The DNA band is collected from the centrifuge tube, extracted with isopropanol to remove the ethidium bromide, and then precipitated with ethanol to recover the DNA. This method allows the isolation of high-quality DNA, but is time consuming, labor intensive, and expensive (an ultracentrifuge is required), making it inappropriate for routine use. This method uses toxic chemicals and is also impossible to automate.

Anion-exchange methods

Solid-phase anion-exchange chromatography is based on the interaction between the negatively charged phosphates of the nucleic acid and positively charged surface molecules on the substrate. DNA binds to the substrate under low-salt conditions, impurities such as RNA, cellular proteins, and metabolites are washed away using medium-salt buffers, and high-quality DNA is eluted using a high-salt buffer. The eluted DNA is recovered by alcohol precipitation, and is suitable for all downstream applications. Anion-exchange technology completely avoids the use of toxic substances, and can be used for different throughput requirements as well as for different scales of purification. The isolated DNA is sized up to 150 kb, with an average length of 50–100 kb.

Silica-based methods — DNeasy Tissue Kits

DNeasy Tissue technology provides a simple, reliable, fast, and inexpensive method for isolation of high-quality DNA. This method is based on the selective adsorption of nucleic acids to a silica-gel membrane in the presence of high concentrations of chaotropic salts. Use of optimized buffers in the lysis procedure ensures that only DNA is adsorbed while cellular proteins, and metabolites remain in solution and are subsequently washed away. This is simpler and more effective than other methods where precipitation or extraction is required. Ready-to-use DNA is then eluted from the silica-gel membrane using a low-salt buffer. No alcohol precipitation is required, and resuspension of the DNA, which is often difficult if the DNA has been over-dried, is not required. DNeasy Tissue Kits are designed for rapid isolation of pure total DNA (genomic, viral, and mitochondrial) from a wide variety of sample sources, including fresh and frozen animal cells and tissues, yeasts, and blood. DNA purified using DNeasy Tissue Kits is free from contamination and enzyme inhibitors and is highly suited for applications such as Southern blotting, PCR, real-time PCR, RAPD, RFLP, and AFLP analyses. DNeasy Tissue Kits are available in convenient spin-column or 96-well formats, suitable for a wide range of throughput needs. Genomic DNA isolated using DNeasy Tissue technology is up to 50 kb in size, with an average length of 20–30 kb. DNA of this length is particularly suitable for PCR analysis as well as Southern blotting analysis. Silica-gel spin technology is not suitable if genomic DNA >50 kb is required for certain cloning or blotting applications. The DNeasy Tissue procedure is suitable for both very small and large sample sizes, from as little as 100 cells up to 5 x 106 cells. In order to obtain optimal DNA yield and quality, it is important not to overload the DNeasy System, as this can lead to significantly lower yields than expected Overloading the DNeasy System can also adversely affect the purity of the DNA The DNeasy Tissue procedure is also highly suited for purification of DNA from very small amounts of starting material. If the sample has less than 5 ng DNA (<10,000 style=""> (a homopolymer such as poly dA, poly dT, or gDNA) should be added to the starting material. Ensure that the carrier DNA does not interfere with the downstream application.

Reference:

www1.qiagen.com/literature/brochures/Gen_DNA_Pur/1019469_BROS_DNYTi_p10_13.pdf

http://bio.classes.ucsc.edu/bio105l/EXERCISES/DNA/genomic.pdf