Utility of amplification enhancers in low copy number DNA analysis
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TECHNICAL NOTE
Utility of amplification enhancers in low copy number DNA analysis Pamela L. Marshall & Jonathan L. King & Bruce Budowle
Received: 8 January 2014 / Accepted: 5 May 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract One parameter that impacts the robustness and reliability of forensic DNA analyses is the amount of template DNA used in the polymerase chain reaction (PCR). With short tandem repeat (STR) typing, low copy number (LCN) DNA samples can present exaggerated stochastic effects during the PCR that result in heterozygote peak height imbalance, allele drop out, and increased stutter. Despite these effects, there has been little progress toward decreasing the formation of stutter products and heterozygote peak imbalance effects during PCR. In an attempt to develop a more robust system that is less refractory to stochastic effects, the PCR additives, betaine, DMSO, PEG, and PCRboost®, were investigated on lowquantity DNA samples. The effects of the additives were assessed by evaluating STR typing results. Of the four additives, the only positive effects were observed with betaine treatment. Betaine, at a final concentration of 1.25 mol/L, was found to improve the robustness of the amplification, specifically by decreasing stutter in a dual locus system. In contrast, the addition of 1.25 mol/L betaine to commercial STR amplification kits did not affect stutter ratios. However, the addition of betaine did lead to increased yield of PCR products in all commercial kits tested. The results support that betaine can improve amplification efficiency of LCN DNA samples. Keywords PCR enhancer . Betaine . Stochastic effects . STR typing . LCN DNA P. L. Marshall (*) : J. L. King : B. Budowle Institute of Applied Genetics, Department of Forensic and Investigative Genetics, University of North Texas Health Science Center, 3500 Camp Bowie Blvd, Fort Worth, TX 76107, USA e-mail: [email protected] B. Budowle Center of Excellence in Genomic Medicine Research (CEGMR), King Abdulaziz University, Jeddah, Saudi Arabia
Introduction The examination of samples with low quantities of template DNA commonly referred to as “low copy number” (LCN) or low template DNA analysis has a major limitation: stochastic effects during the polymerase chain reaction (PCR) are exacerbated, causing heterozygote peak height imbalance, allele drop out, and increased stutter (i.e., artifacts due to slippage during the PCR). All these phenomena can complicate interpretation of LCN profiles. A potential approach to improve robustness of amplification of low template DNA is to modify the PCR by use of additives which effectively concentrate the target and enzyme (i.e., volume excluders), alleviate the paused extension of primer, stabilize the enzyme, and/or reduce instability of the template strand. Robustness of amplification can be measured by reduced stutter values, better heterozygote balance, and increased PCR product yield. A variety of PCR additives and enhancing agents have been the focus of efforts to improve amplificatio
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