Pure and Simple

A General Guide To Prep Sizes DNA Preparation Kits Top Ten Reasons to Try a DNA Preparation Kit DNA is the foundation of life--and of your life's work. The application of many molecular biological techniques in the modern laboratory rests on the ability to isolate pure populations of plasmid DNA and pure, high molecular weight genomic DNA from biological materials. Over the past 10 years or so, clever techniques have been developed for separating the desired DNA component from unwanted RNA, proteins, cellular debris, and chromosomal DNA (in the case of plasmid preps). Most traditional methods of DNA isolation are time consuming, yet they deliver a pure product suitable for virtually any downstream application. However, more and more labs are using DNA purification kits for their speed and convenience. Promega's Wizard Plus SV Minipreps Much of the resistance to time-saving kits involves cost. Since the reagents for the initial steps of DNA purification are very easy and inexpensive to prepare, many cannot justify purchasing kits that provide these same reagents. Also, the purity of kit-prepared DNA has been a major concern for those performing certain downstream applications such as transient transfections; however, the purity obtained by kit methods has improved, and many companies now spell out exactly what "grade" of DNA is prepared from their kits. Purity ranges from "standard" to "ultrapure" and "endotoxin-free ultrapure." What method of DNA preparation can you afford? You must factor in the costs of individual reagents for traditional preparation versus the cost of kits required to perform the number of preps done per week. You must also consider the amount of DNA needed and the amount of time spent waiting in line for the ultracentrifuge. You may be able to cut costs by doing smaller preps--for instance, midipreps rather than maxipreps. Also, what product purity is required? Are you going to make a genomic library or do PCR? Are you going to do transient transfections or subcloning? Is your DNA going to be used in vitro or in vivo? In today's fast-moving research world, obtaining the clone one needs in a few hours versus a few days can make a big difference. In addition, traditional methods of DNA preparation involve hazardous materials requiring special disposal protocols; kits generally use nontoxic reagents and columns that do not require special handling procedures. Today it is hard to find a company that does not sell a plasmid DNA purification or genomic DNA isolation kit of some kind. This article reviews the commonly used methods for DNA isolation to help scientists determine which method best suits their needs. Wizard SV 96 Plasmid DNA Purification System Genomic DNA Isolation The major steps in DNA isolation are cell lysis, deproteination, and recovery of DNA. The main differences between methods concern the enzymes used for deproteination, extent of deproteination, and the desired molecular weight of isolated DNA. An important consideration in choosing genomic DNA preparation methods involves the intended downstream application. For example, if the DNA is to be used simply as a template for PCR, there are a number of speedy methods for preparing samples. Useful products include Bio-Rad's InstaGene™ Matrix, Geno Technologies' OmniTemplate™ and RapidDNA-Template Prep™ Kits, Dynal's Dynabeads DNA DIRECT™ system, and Pierce's Lyse-N-Go™ PCR Reagent. The following sections describe some of the methods available to isolate genomic DNA from various sources. Mammalian Cells and Tissues The traditional method for preparing genomic DNA from tissue is time consuming. Tissue must be rapidly isolated and frozen to prevent DNA degradation by endogenous nucleases, and then crushed into usable pieces. The tissue is then placed in a detergent, sodium dodecyl sulfate (SDS), to solubilize membranes and Proteinase K to degrade protein. The material is deproteinized by phenol/chloroform/isoamyl extractions, and the DNA recovered by ethanol precipitation. For the preparation of DNA for genomic libraries, long high molecular weight (HMW) DNA of 6-25 kb is desirable. In order to obtain such large fragments of DNA, it is important to minimize shearing by gentle but thorough mixing during extraction, and by the use of wide-bore pipette tips. After extraction, organic solvents and salts should be removed by dialysis rather than precipitation. The complete removal of cellular proteins and Proteinase K is important for successful downstream applications (such as restriction enzyme digestion, DNA modifications, etc.). Geno Technologies' MegaLong™ procedure uses a novel device, the Tube-O-Dialyzer™, to minimize sample manipulation (the main cause of DNA shearing) and isolate genomic DNA >100kb from animal tissues, cultured cells, whole blood, yeast, and bacteria. After nuclei are isolated and digested under mild conditions in the Tube-O-Dialyzer, the tube is inverted for dialysis. After dialysis, genomic DNA is ready for use. Genomic DNA isolated from clinical and forensic samples must be free of contaminants and obtained rapidly and efficiently. Many of today's kits are designed to provide high-quality template DNA from blood, blood stains, CSF, and other body fluids for PCR and forensic analysis. Promega's ReadyAmp™ Genomic DNA Purification System is a resin-based system for the isolation of single-stranded genomic DNA from blood and blood stains. DNA isolated using this system may be used directly for PCR without further manipulation. Life Technologies Inc. markets the FTA™ Gene Guard System for convenient collection, transport, and room temperature storage of blood and subsequent DNA preparation for PCR, RFLP analysis, and genomics. This system prepares PCR-ready DNA in less than an hour and is available in card and microwell plate formats. Plants Purifying DNA from plants can be difficult because of the polyphenolic compounds and polysaccharides in plant leaves. For highest-purity genomic DNA containing the nuclear, mitochondrial, and chloroplast genomes, plant cells can be lysed with an ionic detergent (N-laurylsarcosine), treated with a protease, and purified by cesium chloride (CsCl) density gradient centrifugation. Alternatively, plant cells can be lysed with the ionic detergent cetyltrimethyl ammonium bromide (CTAB), which forms an insoluble complex with nucleic acids in a low-salt environment. Polysaccharides, phenolic compounds, and other contaminants remain in the supernatant under these conditions and can be washed away. Precipitated DNA is solubilized by raising the salt concentration and concentrated by an ethanol or isopropanol precipitation. Several companies make genomic DNA preparation kits specifically for use with plant material. These products include Amersham-Pharmacia's Nucleon PhytoPure system, Clontech's NucleoSpin® Plant Kit, Epicentre Technologies' MasterPure™ Plant Leaf DNA Purification Kit, Molecular Research Center's DNAzol® ES reagent, BIO 101's Floraclean™, and QIAGEN's DNeasy™ Plant Kit. Some standard genomic DNA purification kits also efficiently isolate genomic DNA from plant material. QIAGEN's anion exchange technology. Bacteria The bacterial cell wall must be scaled when purifying genomic DNA from bacteria. The main component of bacterial cell walls is peptidoglycan, which is made up of a backbone of repeating disaccharides. Each disaccharide is joined to a tetrapeptide of alternating L and D amino acids, and peptide bridges linking the C terminus of one tetrapeptide to the NH2 group of a tetrapeptide on a neighboring glycan chain. The walls of gram-positive bacteria also contain teichoic acids (long chain polymers of glycerol or ribitol) that protrude from the surface. For traditional bacterial DNA extraction, cells are lysed osmotically or by treatment with detergent (SDS). Polysaccharide and residual proteins are removed by CTAB precipitation and nucleic acids recovered by isopropanol precipitation. CTAB is then removed by an ethanol wash. For large-scale preparation, the process is scaled up and combined with CsCl density gradient ultracentrifugation. Several genomic DNA purification kits can be used to prepare genomic DNA from bacteria. Some of these kits include Clontech's NucleoBond® Tissue Kit, Epicentre's MasterPure DNA Purification Kit, Geno Technologies' OmniPrep™ and MegaLong™ kits, Promega's Wizard® Genomic DNA Purification Kit, QIAGEN's DNeasy kit, and Sigma's Genomic DNA Isolation Kit. Yeast Fungi grow either as single cells (yeast) or as multicellular filamentous colonies (molds and mushrooms). In many molds and yeast, the main cell wall component is chitin (also found in exoskeletons of crustaceans). Yeasts also contain an insoluble glucan, made of ß-1,6-linked D-glucose residues with ß-1,3-linked branches at frequent intervals, and a soluble mannan, an *-1,6-linked polymer of D-mannose with *-1,2 and *-1,3 branches. Yeast experiments often require the purification of both plasmid and chromosomal yeast DNA. Plasmid DNA (generally shuttle vectors) is often used for transformation of E. coli and chromosomal DNA for Southern blot analysis, PCR, or cloning of an integrated plasmid. The most commonly used protocol has been dubbed "smash and grab." Cells are spun down, resuspended in an SDS and Triton "breaking buffer" along with glass beads and phenol/chloroform/isoamyl alcohol. The mix is vortexed vigorously, and plasmid DNA retreats to the aqueous layer. For obtaining chromosomal DNA, this procedure is scaled up with additional TE added prior to vortexing. The DNA in the aqueous layer is ethanol precipitated, resuspended, and treated with RNase A. Several companies make genomic DNA preparation kits specifically for yeast. These products include CPG Inc.'s DNA-Pure™ Yeast Genomic Kit, Epicentre Technologies' MasterPure Yeast DNA Purification Kit, and Zymo Research's YeaStar Genomic DNA Kit™. Some standard genomic DNA preparation kits also efficiently isolate yeast genomic DNA. QIAGEN's silica-gel membrane products. Plasmid DNA Preparation Isolation of plasmid DNA from bacterial cultures may be the most ubiquitous molecular biology technique around. Among other applications, it is essential for analysis of recombinant clones. Typically miniprep procedures are used for rapid screening or rapidly gathering sequence information from cloning experiments. The starting culture volumes range from 1-5 ml with a yield of 5-20 µg of plasmid DNA. When greater amounts of plasmid DNA are required, larger-scale preparation kits are available (midi-, maxi-, mega-, and gigapreps). The first step in any plasmid prep is crude lysate preparation, and most kits supply the necessary reagents. However, some companies sell columns or resins separately, allowing users to prepare their own lysis reagents. Three different methods are typically used to obtain crude lysates for minipreps: alkaline lysis (generally with SDS and NaOH), boiling, or lithium chloride treatment. Most kits utilize the alkaline lysis method. At high pH and in the presence of SDS, cells are lysed and both genomic and plasmid DNA are released and denatured. Bacterial cell wall material and most of the cellular proteins are also denatured during alkaline lysis. When the lysate is neutralized with potassium acetate, the SDS precipitates and the potassium salt and most of the plasmid DNA go into solution. Genomic DNA remains associated with the insoluble cellular debris. The insoluble matter is then removed by centrifugation, and the resulting cleared lysate contains most of the plasmid DNA. The traditional next step is to extract the supernatant with phenol/chloroform/isoamyl alcohol and ethanol precipitate the aqueous layer containing the plasmid DNA. RNase treatment of the lysate or the final resuspended plasmid DNA is often the finishing touch. For larger-scale preps, plasmid DNA is traditionally purified by CsCl density gradient ultracentrifugation, which is the "gold standard" for most researchers. However, the incorporation of resins and spin column chromatography into plasmid purification has freed researchers from lengthy ultracentrifuge spins. The lysate itself is treated with RNase to remove contaminating RNA and a spin column or DNA binding resin is used for removing purified DNA from the supernatant. So what are the matrices and resins that make these kits so expedient? Most kits use silica-based resins alone or coupled with positively charged moieties to allow anion exchange. Spin columns have increased the speed and throughput for many researchers. With some commercial kits, high-quality DNA rivaling that obtained by CsCl gradient purification can be prepared in less than a day. In addition, kits do not require the use of toxic or mutagenic substances, such as phenol, chloroform, or ethidium bromide, eliminating exposure and disposal concerns. Silica Technology The binding of DNA to silica matrices has been a time saver for many researchers. For example, GLASSMILK® from BIO 101 is a silica matrix that binds nucleic acids rapidly, quantitatively, and specifically. Glass binding and elution properties vary with pH and salt concentration; low pH and high salt promote binding while dissociation of the bound nucleic acid occurs at high pH and low salt concentration. The downside to using silica resins or slurries is that they can carry over and interfere with downstream applications and be a potential source of endotoxin contamination. However, the incorporation of silica on columns minimizes these problems. Nucleic acids are adsorbed to the silica gel membrane in the presence of high concentrations of chaotropic salts, and unwanted cellular debris, polysaccharides, and proteins are spun through. After washing, nucleic acids are eluted in small volumes of low-salt buffer, ready for use. Another silica-based technology used in products such as Bio-Rad's Quantum Prep plasmid purification kits makes use of diatomaceous earth. The unique DNA-binding matrix in these kits is made up of the SiO2-rich exoskeletons of diatoms, the highly porous nature of which increases surface area available for DNA binding. Anion Exchange Highly pure plasmid DNA can be isolated using anion exchange technology. Anion exchange resins are positively charged and typically used in a column format. The cleared lysate is loaded onto the column, and DNA binds tightly to the resin. After a washing step, DNA is eluted from the column with a high-salt buffer. Anion exchange-based purification kits offered by QIAGEN utilize a silica-based resin densely coupled with DEAE groups. In low-salt buffers, negatively charged phosphate groups on the DNA backbone interact with the positively charged DEAE groups on the column resin. After elution and alcohol precipitation, DNA is ready for use. QIAGEN's EndoFree™ Plasmid Maxi, Mega and Giga Kits also incorporate an endotoxin removal step into the plasmid purification procedure for the most demanding downstream applications. With all of these choices, researchers looking to save time and headaches are sure to find a DNA purification kit that suits their needs. The accompanying table lists kits available for genomic and plasmid DNA preparation, and a visit to any company's Web site will provide specific information on each product. Grace B. Athas can be contacted at gpathas@mailhost.tcs.tulane.edu. For More Information

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