The Quest For Pure DNA: Genomic DNA: The Root Of All Molecular Genetic Research

Date: July 6, 1998Genomic DNA Isolation Kits The first isolation of DNA is generally credited to Friedrich Miescher, who in the late 1860s was studying proteins, which were discovered a mere three decades earlier. Proteins and DNA--what's the relationship? While attempting to investigate the characteristics of protein in pus cells, Miescher noticed that weakly alkaline cellular extracts, when neutralized, yielded a precipitate; however, the precipitate did not exhibit any of the then known properties of proteins, limited, though they were. Through further investigation, he demonstrated that the precipitable material originated from nuclei and was present in every cell type he examined. Although he published his findings in 1869, he made very little inroads into further purifying this novel substance, which he termed nuclein. Miescher noted that "[the] complete extraction of cells with hydrochloric acid is very difficult. Several weeks go by until everything goes into solution. The liquids do not settle and they filter very poorly; in brief, I was able to make no progress." (from J. Lederberg, Physiological Review, 23:403-30, 1952). The science of nucleic acid biochemistry, structure, function, and purification has progressed immensely from these early years, hastened especially over the last dozen years by the advent of molecular biology. The goal of DNA purification technology is to produce a homogeneous preparation of DNA roughly 40-150 Kb in length, representing the complete repertoire of genetic information contained within a cell. The two most common obstacles to this goal are hydrodynamic shearing during the physical aspects of processing and nonspecific nuclease digestion. The "classical" technique for DNA purification, used for over 40 years, involves homogenization of the biological source in a protein-denaturing environment, crude isolation of nucleic acids, and then purification of DNA by ultracentrifugation in a dense cesium chloride gradient. Today's plethora of nucleic acid purification technologies includes a combination of techniques: cell/nuclei isolation and lysis, protein degradation (especially with regard to the inactivation-destruction of nucleases), and nucleic acid precipitation and/or fractionation, involving a number of reagents and enzymes. The major advantage of these newer technologies over the "classical" technique is that they circumvent the potential shearing forces inherent in density gradient equilibrium sedimentation. In addition, the inclusion of novel ribonuclease inhibitors and protein denaturants minimizes non-specific DNA degradation while maximizing the degradation of RNA and protein contaminants respectively. Modern DNA purification technologies have advanced to the point that specialized techniques and products have also been developed for DNA purification from specific cell types and tissues. In modern technologies, cell lysis and protein denaturation are accomplished simultaneously, using a combination of proteolytic enzymes (for example, lysozyme, Proteinase K, zymolase), chemical denaturants (for example, sodium dodecyl sulfate [SDS], guanidium isothiocyanate, phenol, chloroform, Triton X-100), and heat. DNA is then precipitated out of solution using alcohol (isopropanol, ethanol) and/or further fractionated by reversible binding to an affinity matrix. Or in a novel twist to the common protocols, non-DNA cellular constituents are precipitated (using cetyltrimethylammoniumbromide [CTAB]) and removed from the DNA-containing extract, then the pure DNA is subsequently precipitated. For a comprehensive discussion of common DNA isolation techniques, consult Current Protocols in Molecular Biology, vol. 1 (F.M. Ausubel et al., eds., John Wiley & Sons, Inc., 1995) or Laboratory Techniques in Biochemistry and Molecular Biology: Hydridization with Nucleic Acid Probes, vol. 24 (P. Tijssen, ier, 1993). The classical technique can take from 3 to over 24 hours to complete and requires much effort, lots of materials (many caustic, toxic, and expensive), and an inordinate amount of the equipment resources of a research laboratory or department. In addition, it is generally geared toward purifying more DNA than is routinely needed in today's labs--labs that are more often interested in purifying small amounts of DNA from hundreds or thousands of samples. Modern protocols, many of them using proprietary matrices and reagents and bundled into kits, can purify DNA in as little as 20-30 minutes, may not require any hazardous reagents, and utilize nothing more than basic laboratory equipment. Additionally, the scale and minimal resources required facilitate the simultaneous and/or automated purification of numerous samples in a single cycle. Key in the development of these new techniques has been the advent of chromatographic technology and the development of novel reagents that take full advantage of the distinct physicochemical properties of DNA and the major contaminants (proteins, lipids, complex carbohydrates, and pigments). DNA binding to a DNA binding matrix has several advantages over standard techniques. Two of the most obvious are that immobilized DNA is not a very good substrate, if it is at all, for nucleases, and it is relatively resistant to physical shearing. Most of these affinity matrices are packaged into microcentrifuge tubes or gravity flow minicolumns, which facilitates use and the ability to process many samples simultaneously. DNA-binding matrices in solution (unpackaged) are also available. Their main disadvantage is that they require a few extra handling steps, but they have an added advantage in that researchers can customize their purification for very small sample sizes and/or small yield requirements. DNA isolation may also be achieved using new proprietary reagent formulations, which can potentially reduce the purification to a simple, two-step process: (1) degrading and washing away non-DNA contaminants, followed by (2) precipitating the DNA. An attractive feature of many of these products is that they are packaged such that researchers can use kits scaled to the amount of purified genomic DNA they require and/or the number of samples they need to process. For example, QIAGEN offers QIAamp genomic DNA purification systems in a variety of formats--a microfuge spin column, a conical centrifuge tube, and a 96-well microplate--and Amersham Pharmacia Biotech offers Micro and Macro formats of the company's RapidPrep Genomic DNA Isolation Kit for blood. In addition, a gravity flow column format is available from QIAGEN for the isolation of genomic DNA up to 150 kB in length, at three different yield ranges. Similarly, the products and protocols of other manufacturers (for example, DNAzol from Life Technologies and ProCipitate from LigoChem) can be scaled by researchers for their particular sample size and yield requirements. These new biotechnology products and procedures for genomic DNA isolation provide modern scientists with another set of tools to add to their tool chest, which should also include tools for plasmid DNA purification and mRNA purification, reviewed in the November 24 and August 18, 1997, and May 14, 1998 and March 31, 1997 issues of LabConsumer, respectively. Point your favorite web browser to http://www.the-scientist.library.upenn.edu/tools.html to view archived copies of these reviews. Armed with these research tools, biotechnology researchers are better equipped to meet the demands of the modern research environment. Incidentally, it was in 1871 that Miescher reported the first use of fish (salmon) sperm as an excellent source of nuclein (DNA), the same material that molecular biologists have used for years as a blocking agent in Southern and Northern blots. For those interested in an insightful and comprehensive historical perspective on the discovery of DNA, I recommend reading A Century of DNA by Franklin H. Portugal and Jack S. Cohen (The MIT Press, Cambridge, Mass., 1977). Christopher M. Smith is a computational molecular biologist in the Center for Advanced Computational Science and Engineering at UCSD and can be reached at csmith@sdsc.edu. Genomic DNA Isolation Kits

The Quest For Pure DNA: Genomic DNA: The Root Of All Molecular Genetic Research

Jul 5, 1998

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