Multiplex Amplification and Typing of DNA Extracted from Various Substrates
Susie DelRio, Amy Hayden, Dawn Jarvis, Deborah DiPierro and Kevin C. McElfresh
The Bode Technology Group, Inc., Sterling, VA
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PCR-based DNA identification methods, specifically short tandem repeat (STR) polymorphisms, have become a fundamental tool for forensic use for two important reasons. First, the amount of template DNA needed to generate an identification is minuscule compared to non-PCR methods. Second, the STR loci currently used for identification testing have high discrimination power, something that is missing from other PCR-based identification methods. Further, STR reactions can also be multiplexed; that is, multiple loci can be assayed simultaneously and in the same reaction mix. Given the small amount of template DNA needed for PCR and the ability to multiplex STR reactions, it is easy to see why STR tests have become a standard method. Due to the amount of data that can be generated per unit time, we have been using multiplex reactions routinely. However, multiplex reactions require careful attention to the optimization of the reaction conditions, e.g. annealing temperature, Mg++ concentration, template purity, and template concentration. Not surprisingly, the critical factors affecting the ability to perform successful PCR reactions are the quality and the concentration of the template DNA.
Validation studies were conducted to evaluate the ability to multiplex the DNA isolated from various substrates including wood, paper, nylon, rayon, colored felt, blue denim, cotton swabs, concrete, tissue, dirt, semen and hair. DNA was isolated using one of the following methods: Chelex™ 100 resin beads (Biorad), Proteinase K digestion, organic extraction, and non-organic methods such as the Promega Wizard™ kit. When necessary, purified DNAs were concentrated using the Microcon concentrator columns from Amicon. All of the purified and concentrated DNAs were amplified on a Perkin-Elmer 9600, using the Promega GenePrint™ quadruplex fluorescent STR systems: CTTv (CSF1PO, TPOX, TH01 and vWA) and FFFL (FES/FPS, F13A01, F13B and LPL). The products were separated on 6% acrylamide gels and analyzed with the Hitachi FMBIO
®-100.Standard protocols were developed for each of the substrates and the DNA isolation methods. Clearly, the quantity and quality of DNA isolated from each substrate depended on the method used to purify the DNA. Therefore, each isolation method and substrate was carefully analyzed and adapted to generate the most efficient yield of high quality DNA. In this instance, quality was judged as a function of the ability to perform PCR on the purified product. For example, using the Promega Wizard™ kits with whole blood, we found that substituting 0.01 M Tris (pH 8.0) for the resuspension buffer provided with the kit consistently resulted in PCR ready DNA. We established the conditions for the amplification of the DNA using fluorescently tagged primers on a PE 9600 that did not require tin foil and oil. By using programmed ramp times, we were able to routinely generate excellent results that were easy to analyze on the Hitachi FMBIO
®-100. In all cases, the quality of the final result, compared with known standards (e.g. K562 DNA) and the consistency of the results, were the benchmarks used to establish the validity of the protocols developed.
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