Technology Makes DNA Isolation, Purification Simple And Swift

WIZARD SERIES: Promega Corp. offers a variety of DNA purification products. One of the first steps that biologists perform in the quest to identify a gene, learn the function of a particular protein, or identify the perpetrator of a violent crime is to purify the genetic material away from other cellular constituents. Where once purifications were nearly as complicated as the biophysical assays performed on the products, now they are in many cases the simplest part of the experiment. While many scientists continue to isolate DNA from their biological samples by methods that are tried and true, products for the simple and swift isolation of both DNA and RNA from sources as diverse as bacteria and human cells abound. Scientists have numerous reasons for wanting to purify DNA. Molecular biologists use cloned genes to examine how regulatory proteins interact with specific regions in DNA, for example, or to create mutant proteins for structure/function studies. Cell biologists require purified DNA and RNA to learn more about the patterns of gene expression during development, or for detecting cytogenetic abnormalities in cells taken from cancerous tissues. Evolutionary biologists, who once resigned themselves to classifying organisms according to how they looked, now delve into the molecular sequences of highly conserved regions of DNA taken from diverse creatures to predict evolutionary relationships among all life. Joseph Solvo, an adjunct professor in the department of microbiology, immunology, and molecular genetics at Albany Medical College, has isolated DNA that is thousands of years old from mummies found in Chile. "We wanted to determine if it was possible to isolate high-molecular-weight DNA from these mummies and then amplify specific sequences by PCR [polymerase chain reaction]," he explains. The regions that Solvo and his associates amplified, with high efficiency, were sequences of highly conserved ribosomal RNA genes. "To use PCR to amplify DNA sequences that are highly conserved, you have to start with DNA that is pure and free of contaminating substance from other organisms that might have associated with the body after the person died." Purifying nucleic acids from a biological sample involves first gaining access to the molecules by opening up cells, and then separating the DNA or RNA away from the other macromolecules also found abundantly inside. "Historically, DNA has been isolated by first lysing cells with a detergent or another method, and then using organic chemicals such as phenol or phenol/chloroform to separate proteins from the DNA," according to Mark Reichardt, technical assistance specialist for molecular biology at Pierce Chemical Company in Rockford, Ill. "This method works well with bacteria, mammalian cells, and plant cells, although researchers may alter their procedure to obtain DNA from a specific organism." At Worthington Biochemical Corp. in Freehold, N.J., a company that specializes in the commercial preparation of biochemicals--including DNA--from a variety of sources, nucleic acids are purified according to traditional methods. Worthington scientists prepare their calf thymus DNA, for example, using a protocol first published in 1957 (S. Zamenhof in S.P. Colowick and N.O. Kaplan, eds., Methods in Enzymology, Vol. III, New York, Academic Press, 1957). "Although the original procedure has been modified and perfected, we find that this method still gives us the best yield of high-quality DNA with lower protein and RNA contamination than most other commercial preparations," maintains Joanne Soderman, a Worthington technical services representative. "For many applications, we still use DNA-purification methods such as cesium chloride centrifugation that consistently provides us with good yields of clean DNA," agrees Richard Zitomer, a professor of biology at the State University of New York, Albany. "In some cases, though, such as purifying DNA fragments out of agarose gels, it is more appropriate to use alternative methods that take less time." KEEPING PACE: Beckman's P/ACE 5510 system with Gold Work Station. "Within the last five years, there has been a shift away from the alkaline lysis and organic extraction techniques used over the last decade, to binding DNA to a glass matrix or similar material to isolate it away from contaminating proteins and RNA," says Reichardt. This technology has become popular because the purification steps are greatly simplified, and in many cases the product is cleaner than DNA prepared using older techniques. "This type of DNA purification procedure is very fast, and it provides us with very clean DNA," notes Zitomer. "And, since there are no precipitation steps, you can do it with limiting amounts of DNA." A typical procedure for isolating plasmid DNA from bacterial cultures can take anywhere from a couple of hours to a few days, depending on the desired level of purity and the skill and experience of the person preparing the DNA. At the end of the prep, the plasmid DNA is theoretically purified away from the bacterial proteins. Unless the DNA is centrifuged in a cesium chloride gradient, RNA from the bacterial cells and possibly even proteins remain. For most applications, with the exception of gel electrophoresis, preparations of plasmid DNA must be free of residual RNA and protein. Therefore, conventionally prepared plasmid-prep DNA must be subjected to additional enzyme treatments and extraction steps before it can be classified as "pure." On the other hand, purification procedures that include a matrix material, such as silica, cellulose, or latex, are accomplished in less time and require far fewer steps. After the cells are lysed, the preparation is added to the support material, to which the DNA or RNA usually binds. Once the nucleic acid is immobilized on the matrix, which is usually packed into a column or cartridge, the contaminating macromolecules can be washed through the column and discarded. The DNA is eluted off of the matrix and collected in water or buffer in one step, without the need for any additional extraction or precipitation steps. The end product is highly purified DNA that is ready to use. "The entire procedure can be accomplished in under an hour," says Zitomer. "Researchers choose a method for DNA purification depending on the application and on how quickly they want the DNA," says Cheryl Helton, technical services scientist for Boehringer Mannheim Biochemicals in Indianapolis. "Generally, column purifications are quicker and a little bit easier to do." Several companies market plasmid DNA purification kits that require only a microcentrifuge or a vacuum apparatus to push (or pull) the reagents through the column. Pharmacia Biotech Inc. of Piscataway, N.J.; Invitrogen Corp. in San Diego; Qiagen Inc., based in Chatsworth, Calif.; and Life Technologies of Gaithersburg, Md., along with Pierce Chemical Corp. and others, supply researchers with columns and reagents required for the complete separation of plasmid DNA from bacterial cultures of all sizes, from 1 ml or 2 ml mini-preps to large-scale preps yielding milligram quantities. A twist on the DNA purification theme takes the form of magnetic beads to which nucleic acids can bind. Using products from Cambridge, Mass.-based PerSeptive Diagnostics Inc. or from Dynal Inc. of Lake Success, N.Y., molecules of DNA are reversibly bound to a support matrix consisting of magnetic particles. The DNA-bound beads can be separated from a cellular extract with the help of magnets that attract the particles, drawing the nucleic acids away from other cellular macromolecules in solution. The DNA is eluted from the beads and the two go their separate ways once again via use of the magnet. For those scientists appreciative of automation, Pharmacia markets a semi-automated system called the EasyPrep, which adds a positive pressure pump to the purification procedure. The pump takes care of the steps in which reagents are passed through the support matrix. According to Pharmacia, up to 24 separate bacterial cultures containing plasmid DNA can be processed in parallel in 30 to 60 minutes. Purification is generally not the only thing a researcher wants to do with plasmid DNA. Once the DNA from a particular plasmid has been isolated, it may be subjected to several other procedures involving, for instance, amplifying sequences by PCR, mutagenesis, or simply cleavage with restriction enzymes for additional subcloning. All of these applications require DNA that is free from contaminating substances like proteins or salts, which might interfere with or even prevent the desired reaction. "For really quantitative studies involving DNA, it's important to know what you are working with," says Zitomer. DNA prepared from resin-based kits is considered clean enough to be used directly in applications like sequencing and PCR that require highly purified DNA. ALL PACKED UP: Glen Research Copr. offers Polypack cartridges. An additional advantage to researchers using column chromatography methods is a reduction in the hazardous chemicals required to achieve a purified product. Chloroform and phenol, two of the standard organics used for nucleic acid extractions, are both toxic and difficult to store. Cesium chloride and ethidium bromide, two suspected carcinogens, are also used in plasmid purifications based on alkaline lysis and nucleic acid precipitation techniques. According to Terry Charmichael, technical service representative for Qiagen Inc., "DNA purifications can be accomplished without the use of organics or other toxic chemicals." Most of the companies that produce plasmid purification kits have extended their technologies to encompass all of the different types of DNA or RNA that researchers might want to purify. Kits in Madison, Wis.-based Promega Corp.'s Wizard series--which are also available through Pittsburgh-based Fisher Scientific under the same brand name--consist of several products that can be used to purify genomic DNA from a wide range of cell types, including blood cells; to purify oligonucleotides away from contaminants after synthesis on a machine; and to clean up amplified fragments generated by PCR. With Pierce's XTreme purification kits, investigators can purify genomic DNA from bacteria, plant, or mammalian cells; quickly separate oligonucleotides from contaminating sequences and salts; and isolate DNA from samples of whole blood. "Our kits designed for the purification of DNA from blood samples use a slightly different technique, in which the proteins actually bind to the silica matrix while the DNA remains in solution," explains Reichardt of Pierce. Pharmacia, Boehringer Mannheim, Invitrogen, and Qiagen also market kits that are suited for specific separations. In some respects, PCR has revolutionized the way many scientists approach their experiments. Where once it was necessary to purify sufficient amounts of DNA to use as starting material for cloning experiments or genetic analysis, now only a few molecules are required. With PCR, specific sequences targeted by oligonucleotide probes are replicated over and over by a heat-stable polymerase, thus amplifying the target sequence millions of times. The fragments generated by PCR can be cloned, sequenced, or analyzed for restriction fragment-length polymorphisms (RFLP). One caveat is that the starting material for PCR should be pure and of high quality. Biotech manufacturers have responded to this requirement by developing products that are specifically designed for isolating DNA from samples of blood or body tissues. The Split Second DNA isolation kit by Boehringer Mann-heim, the XTreme Genomic DNA purification kit from Pierce Chemicals, and the Easy-DNA kit from Invitrogen are examples of products specifically geared toward isolating genomic DNA intact from whole blood. But forensic samples don't always make it to the laboratory in the form of freshly drawn whole blood. The Readyamp Genomic DNA-purification kit by Promega is a system for isolating DNA from bloodstains or from samples of dried blood. In either case, the DNA product is clean enough to take directly into a PCR reaction. There are additional purification problems associated with PCR. The nucleic acid that acts as a template for the amplifying rounds of DNA synthesis must be kept free of contaminants. This means that DNAs from other sources must be kept out of the sample. To maximize the amplification reactions, it is advantageous to keep the DNA separate from the other reagents until the optimal annealing temperature is reached. Molecular BioProducts Inc. of San Diego has developed two products that specifically address these aspects of PCR. The company's ART Barrier pipet tips contain a barrier material that prevents both liquids and aerosols from passing through. When placed on the end of a micropipettor, ART tips protect the pipettor from contaminating DNA. Molecular BioProducts' HotStart reaction tubes for PCR contain a minute wax bead that comes pre-adhered to the interior surface of the tube. The wax forms a physical barrier that separates the template and polymerase from the oligonucleotide primer until the optimal annealing temperature is reached. During thermal cycling, the wax melts and the reagents mingle, thus priming the PCR reaction. Hot-starting PCR reactions improves the specificity of the reaction by reducing the number of false primes. After a particular DNA sequence has been amplified, the PCR products are often separated by electrophoresis through an agarose gel. From there, individual fragments can be visualized and then extracted to be cloned or further analyzed. Separating fragments in this way is usually the preferred method, because it allows investigators to identify specific fragments by their molecular size. "Generally, we run our PCR-generated fragments on a gel to double-check the DNA, then cut out the right-sized fragment and clone it," explains Zitomer. However, once the DNA has wormed its way through the network of pores in an agarose matrix, it's not always that simple to get it back out. Several products allow researchers to separate small pieces of DNA, such as fragments in agarose gels and oligonucleotides after DNA-synthesis reactions. NACS-52 Prepac cartridges from Life Technologies and the QIAquick kit from Qiagen are but two examples. Polypack cartridges from Glen Research Corp. in Sterling, Va., are specifically designed for small-scale purifications of oligonucleotide products that have been artificially created on a DNA synthesizer. Once an oligonucleotide has been synthesized, protecting groups--chemical moieties that block uninvolved reactive sites--are removed with ammonium hydroxide treatment, leaving a dimethoxytrityl protecting group on one end of the molecule. Notes Bob Somers, senior applications chemist at Glen Research Corp., "This highly polar protecting group allows the full-length oligonucleotides to be separated from the failure sequences by reverse-phase chromatography in the Polypack cartridge." Somers adds that his company's cartridges are designed for bulk purifications of small amounts of DNA. "For larger- scale purifications of oligonucleotides, HPLC [high- performance liquid chromatography] is the best method," he notes. "For companies trying to develop DNA-based drugs and synthesize them in large quantities, a combination of reverse-phase chromatography and ion-exchange chromatography using an HPLC would allow for separations of oligonucleotides in sufficient quantities and with the proper level of purity." The P/ACE series of capillary electrophoresis systems from Beckman Instruments Inc. of Fullerton, Calif., performs high-resolution analysis of single-stranded oligonucleotide primers, probes, and antisense drugs, as well as the double- stranded products of PCR reactions. Capillary electrophoresis can be used for fragment pattern analysis (RFLP) for DNA fragments smaller than 1,000 base pairs. When it comes to deciding which DNA separation technology to use, investigators must consider their application. "There are pluses and minuses associated with all of the various technologies," concludes Helton of Boehringer Mannheim. "It all depends on what you're trying to do." Holly Ahern is a science writer and an assistant professor of biology at Adirondack Community College in Queensbury, N.Y.

Technology Makes DNA Isolation, Purification Simple And Swift

Interested in reading more?

Become a Member of