Get the Gel Out of Here

Date: March 29, 1999Fragment Purification Products Table Purifying DNA fragments from gels is one of those chores that no one likes to talk about--it's not glamorous, yet it is essential for a functioning molecular biology lab. There are almost as many methods around for doing this as there are researchers. And everyone swears by his or her favorite method. The fact is that probably all methods work to some degree, and most work reasonably well. There are two basic approaches to isolating fragments, electrophoretic and chemical, and to some extent, it's really a matter of taste in choosing between the two--whether you like to tinker with an electrophoresis apparatus or whether you would rather work in a test tube. For purification of PCR fragments, the more recent advances in this methodology avoid running gels at all, and of course, high throughput systems are becoming more common every day. PCR products purified with ArrayIt™ Human cDNA inserts were amplified by PCR and purified with an ArrayIt™ 96-Well PCR Purification Kit. 10% of each sample (lanes 2-13), together with molecular weight markers (lane 1), was fractionated on 0.8% agarose, stained with ethidium bromide, and photographed. Fluorescent image is represented in a pseudo color scale. Image Provided by Telechem International Electrophoresis can be harnessed to purify fragments in two ways: (1) following resolution on gels, fragments can be trapped onto a membrane inserted in the gel just ahead of the fragment (band interception method) or (2) fragments can be electroeluted out of an excised piece of gel. Electroelution can be done simply by dropping the gel slice into dialysis tubing with a little buffer and placing it back in the electrophoresis apparatus. Alternatively, devices both simple and complex can be used for this. On the simple end are GeneCapsules, invented and sold by GenoTechnology and offered by Stratagene as Quik-Pic Electroelution Capsules. Gel slices are put into these chambers and, as with dialysis membranes, returned to the electrophoresis chamber for a few minutes to move the DNA out of the gel into the surrounding solution. Some rather complex devices also are available for electroeluting DNA from Schleicher and Schuell (Elu-Trap), Millipore (Centrilutor Micro-Electroeluter), and BioWorld (Electro-Elutor-Dialyser-Concentrator). While these devices are more elaborate and expensive, they have multiple uses: they can be used for proteins as well as DNA and for desalting, concentrating, and eluting macromolecules. At the top of the line, at least for plasmid purification, is the MacConnell "Time Machine", which goes from bacterial colony to pure, electroeluted plasmid DNA without the touch of a human hand. While electroelution was among the earliest methods for isolating fragments, supplanted in recent years by the chemical or enzymatic methods, it is experiencing a comeback--particularly for the isolation of very large molecular weight fragments used in artificial chromosome construction. In this instance, using electroelution avoids the use of enzymes and procedures that could compromise the integrity of the DNA.1 In addition, unlike some of the kits to be described later that require working with low melting point (LMP) agarose, electroelution can be done with regular agarose (and other matrices, such as acrylamide, for that matter), which provides better resolution and is easier to handle than LMP agarose. When working with lots of samples, or if you're just plain tired of fiddling with dialysis tubing or membranes, you might want to consider one of the reagents or kits that can be used to extract DNA from gel slices. Some require LMP agarose, while some can be used with any type of agarose. Some kits work only with TAE buffer, while other kits provide modifying buffers so they can also be used with gels run in TBE. One advantage of many of these kits is that they are multifunctional and can also capture or purify DNA from solutions and reactions. Since most formats have a lower limit of from 40 to several hundred base pairs, they can be used to isolate PCR fragments from primers and primer-dimers, obviating the need to run out PCR reactions on gels. Among the simplest methods for extracting DNA from gel slices is the so-called squeeze freeze method. In this method, the gel is first made brittle by freezing and then crushed by mechanical force provided by centrifugation, squeezing out the buffer containing DNA into a collection tube. The only trick here is cutting--the smaller the size piece of gel that is cut, the smaller the buffer volume of the resulting isolated fragment (that is, the higher the DNA concentration). According to Bio-Rad, which provides a kit for this method, DNA so purified can be used in all downstream applications, including automated fluorescent sequencing. Most kits are based on the property of silica to bind DNA in the presence of high salt or chaotropic agents.2 The silica method is convenient since (1) most contaminating molecules, large and small, don't bind, (2) it can be used with gel slices or with DNA in solution, regardless of the salt concentration, and (3) the DNA is eluted in low ionic strength buffers, often requiring no further purification or even concentration. Some kits contain silica in solution (glassmilk), while others have silica embedded in pads or columns that can be vacuum filtered, spun, or both. Spin columns and vacuum formats are easier to use than solution-based silica, but are not scalable. Some companies have modified the silica in some way to make it either more reproducible or easier to resuspend, which can improve yields. And then there is agarase, which simply enzymatically digests the polysaccharide backbone of molten agarose to soluble monomers and oligosaccharides, available from a number of suppliers. Longest in the market with an agarase, Epicentre offers GELase™ from an undisclosed source. Most of the rest come from on the market Pseudomonas atlantica. Promega recently starting selling an agarase isolated from a marine flavobacterium under the name ß-AgarACE, The claim is that with agarase you can get complete digestion of agarose, yielding 100 percent of your sample. As with all enzymes, it pays to keep a close eye on unit definition when comparing prices among suppliers. And scale-up is no problem. Epicentre reports that using its high activity protocol, a customer digested 1 kilogram of agarose with 200 units of its enzyme in 10 hours. Recovery of DNA using any of these methods is almost always less than 100 percent, as you'll see in the accompanying table, but these methods tend to be quick, easily scalable, affordable, and adequate for many applications. Some, though, require further organic extractions or purification, which adds to the time and might also affect yield and the integrity of the sample. Some might ask, "Why bother with extracting DNA from gels, as the list of enzymes that work in the presence of agarose is considerable?" Among them (according to some) are alkaline phosphatase, BAL 31 nuclease, DNA polymerase I, Klenow fragment, restriction endonucleases, reverse transcriptases, T4 DNA ligase, T4 DNA polymerase, T4 polynucleotide kinase, T7 DNA ligase, T7 DNA polymerase (Sequenase® polymerase), and Taq DNA polymerase. Some applications of PCR require running rather large numbers of samples, which can be tedious, time consuming, and expensive. Kits that will isolate PCR-amplified fragments directly from the reaction mix are available; these save the time and expense of running gels. They separate primers and primer-dimers from the PCR fragment itself. Accordingly, when choosing a kit or method, attention should be paid to the minimum number of base pairs detained on the filter or matrix to insure that this separation will be successful. Separation of homoduplexes from heteroduplexes under partially denaturing conditions using Transgenomic WAVE DNA Fragment Analysis system In addition, a number of companies have created kits suited for large numbers of samples, particularly for purifying PCR fragments. QIAGEN offers its QIAquick Multiwell PCR Purification kit in 8-well strips and 96-well plates that are vacuum or centrifuge compatible and fully automatable. Promega's Wizard® Series 9600™ Vacuum Manifold handles up to 96 samples, and for really big sample numbers, TeleChem International's ArrayIt™ PCR Purification Kit is geared for 384-well centrifugation format. As the questions being asked and the methodologies being applied in biotechnology get increasingly sophisticated, the purity and quality of materials takes on greater importance. And so it is for some of the uses of isolated DNA fragments. Anyone who has ever run an agarose gel will have no trouble accepting the fact that a band may not be a single DNA species, but rather a collection, albeit a small collection, of similarly-sized molecules. Nonetheless, for some studies, even a small amount of another species can confound the analysis. While gel systems exist that have superior resolution power, they are not always the easiest to work with, particularly in high-throughput situations. Help is at hand. Transgenomic's WAVE™ Fragment Analysis System provides rapid gel-like separation and isolation of DNA fragments (up to 1,000 base pairs) in a non-gel based system. The WAVE uses ion-pair, reverse-phase, high performance liquid chromatography in a fully automated and computerized instrument. This system has been shown to resolve species unresolvable by conventional agarose gel electrophoresis,3 and in a recent study, the WAVE was shown to separate homoduplexes from heteroduplexes4 containing a single base-pair mismatch. The WAVE combines a 96-well autosampler for high throughput screening with its DNASep® separation technology and analysis system (GelPeak® software). Whatever your preference--glass, membranes, electricity or frozen bits of gel--several sources of information are available for perfecting and optimizing your technique. FMC BioProducts (Rockland, ME), maker of a variety of agaroses, has a sourcebook called Your Complete Guide for DNA Separation and Analysis, (available on its Web site www.bioproducts.com) full of detailed protocols and tricks to getting these methods to work. In addition, many of the kit providers have protocols and documentation of their products on their Web sites. S.J. Strong et al., "Marked improvement of PAC and BAC cloning is achieved using electroelution of pulsed-field gel-separated partial digests of genomic DNA," Nucleic Acids Research, 25:3959-61, 1997. B. Vogelstein, D. Gillespie, "Preparative and Analytical Purification of DNA from agarose," Proceedings of the National Academy of Sciences, 76:615-9, 1979. K.H. Hecker et al., "Optimization of Cloning Efficacy by Pre-Cloning DNA Fragment Analysis," Biotechniques, 26:216-22, 1999. A. Kulkin et al., Isolation and Analysis of Amplified cDNA Fragments During Detection of Unknown Polymorphisms with Temperature Modulated Heteroduplex Chromatography. In press. The author can be contacted at ldefrancesco@the-scientist.com Fragment Purification Products Table

Get the Gel Out of Here

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