MadSci Network: Molecular Biology

Re: genomic DNA extraction standarad Dellaporta protocol

Date: Mon Nov 1 14:57:01 2010
Posted By: Jeff Buzby, Ph.D., CHOC Research Institute
Area of science: Molecular Biology
ID: 1284718046.Mb

Dear Anjana:

Based on the 'standard Dellaporta gDNA extraction protocol' that you're interested in (as described in Cold Spring Harbor Protocols), I'll try to briefly explain the main role of each reagent, to the best of my knowledge.

The Tris-HCl, contained in the extraction (EB) as well as the BTE and TE buffers, is the actual pH-regulating buffer system.   It's important to maintain a slightly basic pH for storage of nucleic acids, since they can be subject to acid hydrolytic degradation.  So Tris-HCl is typically used to maintain a pH > 7 for nucleic acids, with 8.0 probably the most common choice for DNA.

EDTA is a chelator of divalent metal ions, which means that it binds very strongly to ions such as Mg+2, Mn+2, or Ca+2 so that they're no longer available for other potential reactions.  Mg+2 happens to be a common cofactor of nucleolytic enzymes that can degrade DNA, so EDTA can help to inhibit these reactions and stabilize the gDNA by sequestering Mg+2 ions.

β-Mercaptoethanol is a very strong (and smelly) reducing agent that unfolds proteins from their natural active conformations by breaking the disulfide linkages between their cystein residues.  This can also help to inactivate endogenous nucleolytic enzymes, but is probably even more useful in decreasing the solubility of the protein impurities so that they precipitate better in the 1st centrifugation step.

The fairly high concentration NaCl (sodium chloride, common salt) in the EB (500 mM) also facilitates protein precipitation.

SDS, or sodium dodecyl sulfate, might be the single most important reagent for DNA extraction. It is a powerful detergent that will both quickly dissolve cell membranes and further denature protein impurities, breaking them down to their individual polypeptide subunits by disrupting their hydrophobic interactions to facilitate their inactivation and precipitation.  So SDS single-handedly helps to remove two of the biggest biological contaminants of nucleic acids - lipids and proteins.

The potassium acetate interacts with the SDS to further facilitate protein precipitation in the 1st centrifugation step.

Sodium acetate, along with the potassium acetate carried over in the supernatant from the 1st centrifugation step, also greatly enhances the yield of DNA precipitated in the following steps.

Both isopropanol and ethanol are very effective DNA precipitants. Ethanol is generally considered to be more effective, creates tighter pellets, and is easier to evaporate afterwards.  But isopropanol is sometimes chosen because it often requires less volume and many protocols prefer it for initial precipitation of gDNA, although the specific rationale isn't clear to me.  70% ethanol is then used to wash away some remaining water soluble impurities without dissolving the gDNA, to 'tighten' the gDNA pellet, and to replace residual isopropanol with ethanol for evaporation.

The step where the 1st isopropanol precipitate is resuspended in an aqueous BTE buffer (very similar to TE buffer) and then centrifuged is apparently to remove any insoluble materials, most likely coprecipitated but less soluble polysaccharides from the plant cell walls.

Finally, RNase A is used to degrade most of any copurified, contaminating RNA.


So here's a shorter summary of the major roles for the most important extraction components:

Cell lysis - SDS

Protein precipitation - SDS, β-mercaptoethanol, NaCl, potassium acetate

DNA precipitation - isopropanol, ethanol, sodium acetate

Polysaccharide precipitation - low volume aqueous (BTE) resuspension

RNA removal - RNase A

DNA stabilization - Tris-HCl, EDTA (TE)

I hope this info. helps you to understand and perform your extractions more efficiently,


Jeff Buzby, Ph.D.
CHOC Research Institute

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