PCR on the Run

PCR Template Preparation Kits Epicentre Technologies' MasterAmp Buccal Swab DNA Extraction Kit Among the thousands of kits and products available to the life scientist, many provide a means of purifying genomic and plasmid DNA.1 Though most of these prepare highly purified DNA for specific applications such as Southern blotting, fluorescent automated sequencing, and RFLP analysis, some are designed to prepare DNA simply for use as PCR templates. Generally characterized as fast, easy, and efficient, these template preparation products move away from the traditional DNA purification techniques including phenol and chloroform extractions and alcohol precipitations. DNA-releasing agents, paramagnetic beads, various resins, and spin columns are typical strategies employed here to quickly prepare PCR-ready DNA from a very broad range of sample materials. The easiest and most direct way to prepare and amplify template DNA is to perform all the necessary steps within a single tube. Two products have been developed with this strategy in mind. The latest, Lyse-N-Go™ PCR Reagent from Pierce Chemical Co. of Rockford, Ill., releases DNA for direct PCR amplification from bacteria, yeast, some animal and plant tissues, whole blood, and cultured mammalian cells in less than 10 minutes. To illustrate, bacterial colonies can be rapidly screened for inserts following transfer of a small amount of each test colony with a pipet tip into 5 µl of Lyse-N-Go Reagent. After a two-minute heating step at 95°C, gene-specific primers and amplification reagents are added to the lysate. Amplification occurs with standard PCR procedures. Lyse-N-Go Reagent is compatible with several commercially available Taq polymerases and has applications in high-throughput screening procedures. CPG Inc. of Lincoln Park, N.J., offers an alternative DNA-releasing reagent for PCR. Known as Release-IT™, this product lyses cells upon heating while sequestering lysis by-products, improving the yield and specificity of amplification. Typically, samples are placed into an amplification tube using a 1:19 µl ratio of sample to Release-IT reagent. Lysis is conducted by heating the tube in a thermal cycler or microwave oven for 10 minutes, and amplification reagents are then added directly to the lysed sample. Release-IT works with just about any biological sample containing nucleic acids. In fact, CPG has developed at least 28 amplification protocols for samples as diverse as goat hair, dental plaque, whole blood, urine, and paraffin-embedded tissue. Multisample Kits Multisample kits prepare PCR-ready DNA templates from a variety of sources, yet require techniques and procedures somewhat more involved than those of the single-tube products. Differing slightly from these products in that the freshly prepared template must be transferred into an amplification reaction tube for PCR, the InstaGene Matrix from Bio-Rad Laboratories of Hercules, Calif., nevertheless provides PCR-quality DNA using a single-tube procedure in about an hour. Consisting of a specially formulated Chelex resin, the InstaGene Matrix absorbs cell lysis products that interfere with PCR. In addition, because the DNA is not bound to any of the purification reagents, the matrix reduces DNA losses due to irreversible binding. In a typical application, the InstaGene Matrix is added to the sample and heated at 56°C for 30 minutes. After a brief high-speed vortexing step, the sample is heated again at 100°C for eight minutes. Following a short centrifugation, the resulting supernatant provides DNA template for transfer into amplification tubes. Protocols are available for preparing DNA templates from whole blood, cultured mammalian cells, and bacteria. Geno Technology of St. Louis offers another single-tube method for preparation of PCR-ready DNA from a host of samples. The OmniTemplate™-DNA Template Prep Kit isolates nuclei in the same tubes in which samples are collected. The isolated nuclei are then digested with Templating Buffer and Proteinase K to release template DNA in a buffer compatible with PCR and other applications. Usually, 5 µl of the purified DNA provides sufficient template for a 50 µl amplification reaction. Protocols for animal tissues, blood, and cell cultures are available. The Rapid DNA-Template Prep™ Kit, also from Geno Technology, is a resin-based product developed to obtain template DNA from blood, animal tissues, cultured cells, and plant and fungal samples. The Rapid DNA-Template Kit uses a proprietary resin called pinkRESIN™ to capture DNA templates in cell lysates. Spin-column washing techniques remove impurities before the template DNA is eluted from the pinkRESIN in a small volume of elution buffer. Roche Molecular Biochemicals of Indianapolis offers the High Pure PCR Template Preparation Kit for preparation of PCR-ready DNA from a variety of sources. For whole blood or cultured cells, the total time required for preparation is only 20 minutes (12 minutes hands-on time). For amplification of viral DNA from serum, plasma, whole blood, or cultured cells, the company offers the High Pure Viral Nucleic Acid Kit. The system allows amplification of sequences from DNA or RNA viruses by PCR or RT-PCR. The High Pure 16 System Viral Nucleic Acid Kit works on the same principle and prepares 16 samples in parallel. Dynal ASA of Oslo, Norway, has applied its superparamagnetic bead technology to simple and rapid purification of PCR-ready DNA from a wide range of sample sources.2 Tackling samples such as cultured cells, bacteria, mouse tails, feces, and algae, the Dynabeads® DNA DIRECT™ System I relies on cell lysis and the subsequent absorption of DNA to the surfaces of the Dynabeads DNA DIRECT paramagnetic particles. The DNA/Dynabead complexes are pulled from solution by applying a magnetic particle concentrator (Dynal MPC®). Supernatants and washing solutions containing impurities and potential inhibitors are removed in the same manner. Finally, the DNA/Dynabead complexes are resuspended and used directly in amplification reactions. Alternatively, the DNA may be eluted by incubation at 65°C. Blood Relations Preparation of DNA templates from blood is a common application for DNA purification kits. A few have been developed to prepare PCR-ready genomic DNA strictly from blood. The S.N.A.P.™ Whole Blood DNA Isolation Kit from Invitrogen Corp. of Carlsbad, Calif., for example, is designed to purify "ultrapure" DNA from whole blood samples between 1 and 1,000 µl. The process employs Invitrogen's exclusive silica-based S.N.A.P. DNA-binding resin bound in a membrane/column format. This spin-column configuration facilitates multiple-sample processing. Typical yields are around 0.5 µg of DNA from 150 µl of blood. The kit works consistently with all fresh, frozen, and dried blood samples. To prepare single-stranded DNA from whole blood or bloodstains, Promega Corp. of Madison, Wis., offers the ReadyAmp™ Genomic DNA Purification System. A resin-based product that also employs a single-tube isolation procedure, the ReadyAmp System produces single-stranded DNA suitable for direct use in amplification reactions in less than an hour. Typically, 1 to 5 µl of the purified single-stranded DNA is enough for a 50 µl amplification reaction. The kit extracts DNA from 1 to 400 µl of blood per sample or from samples of bloodstained material between 9 and 25 mm2. Between 0.04 and 10 µg of DNA can be obtained as the sample volumes increase across the 1 to 400µl range. Dynal has applied the same superparamagnetic bead technology described earlier to simple and rapid purification of PCR-ready DNA from blood, buffy coat, and bone marrow. The Dynabeads DNA DIRECT System II isolates enough DNA from 100 µl of blood to supply at least 100 PCR amplifications in about 15 minutes. The yields depend on the number of nucleated cells in the sample; a 100 µl sample generally produces 1 to 5 µg of DNA. In addition, the Dynabeads DNA DIRECT System II has been used successfully on individuals with white blood cell counts below the normal human range. For automated isolation of PCR-ready DNA from whole blood in a 96-well format, Dynal's Dynabeads DNA DIRECT Auto96 processes 96 samples (10 to 30 µl) in 80 minutes when used in combination with an integrated magnet station (for example, the Dynal MPC-auto96 or similar station) and a liquid-handling robot. Roche Molecular Biochemicals' Split Second™ DNA Preparation Kit is a single-tube method for the preparation of PCR template DNA from human whole blood. The kit consists of two solutions: the first preferentially lyses red blood cells to allow pelleting of intact white blood cells (WBCs) and the second releases the WBC DNA. This month, Stratagene of La Jolla, Calif., introduced the StrataPrep Blood DNA Kit for PCR, which prepares PCR-ready DNA from 25-200 µl of whole blood in about 40 minutes. Each purification requires just one microspin cup placed in a 2 ml collection tube and does not require pelleting of WBCs, protease digestion, or incubations above room temperature. A proprietary membrane in the microspin cup retains WBCs while contaminants are spun through. WBCs are then lysed by addition of a chaotropic salt solution, and released genomic DNA binds to a glass fiber matrix positioned below the membrane. After washing and elution in a small volume, up to 50 PCR amplifications can be performed from a single preparation. A Cheeky Product The MasterAmp™ Buccal Swab DNA Extraction Kit from Epicentre Technologies of Madison, Wis., offers an interesting alternative to extraction of PCR-ready DNA from blood. The purification of DNA from easily obtained buccal (cheek) cells eliminates the expense, patient distress, and special handling procedures required to minimize exposure to bloodborne pathogens associated with blood samples. The noninvasive sampling is easily done by individuals without extensive training, increasing voluntary participation in research studies, among other benefits. For example, one cystic fibrosis screening study estimated that buccal sampling increased participation by 25 percent over blood draws.3 Buccal cells are readily obtained by swabbing the inside of the cheek (human or mammalian) with the kit's buccal brushes. After transfer of the cells to DNA extraction buffer, heat treatment lyses them and degrades any compounds inhibitory to amplification. A centrifugation step removes debris. The DNA is then ready for PCR. In addition, the extracted DNA contains Epicentre's MasterAmp PCR Enhancer, a proprietary reagent designed to improve product yield. The procedure generally yields 0.5 to 3 µg of DNA per buccal sample in about an hour. Pinpoint Accuracy The Pinpoint Slide DNA Isolation System™ from Zymo Research of Orange, Calif., is a unique PCR template preparation product. Designed for use with tissue sections mounted onto microscope slides, this system enables recovery and amplification of DNA from selected areas on the slide. The kit combines Zymo Research's Pinpoint tissue sampling technology with a one-step DNA extraction method. Here, Pinpoint Solution is applied to the selected area on the slide. As the solution dries, the underlying tissue becomes embedded in a thin film. The area is then lifted away from the slide, and the DNA is recovered with a one-step extraction buffer. At this point, the DNA is suitable for PCR analysis. Typical sample sizes are about 1 mm square and 10 µm thick, and consist of 200 to 400 cells. As life science research advances, the enormous complexity of biological pathways is steadily being revealed. Luckily for researchers, tools for studying these problems are rapidly evolving to add speed and convenience to many lab procedures. Kits for the rapid preparation of PCR template DNA are sure to speed many scientific pursuits, pleasing graduate students and advisers alike. Michael D. Brush can be contacted at michaeldbrush@cs.com. References 1. G. Athas, "Pure and simple," The Scientist, 13[22]:20-2, Nov. 8, 1999. 2. A. Deggerdal, F. Larsen, "Rapid isolation of PCR-ready DNA from blood, bone marrow and culture cells based on paramagnetic beads," BioTechniques, 22:554-7, 1997. 3. E.W. Clayton et al., "Lack of interest by nonpregnant couples in population-based cystic fibrosis carrier screening," American Journal of Human Genetics, 58:617, 1996.

PCR on the Run

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