Theresa Caragine

The Application of Ultraviolet Irradiation to Exogenous Sources of DNA in Plasticware and Water for the Amplification of Low Copy Number DNA

ABSTRACT: Using high sensitivity forensic STR polymerase chain reaction (PCR) typing procedures, we have found low concentrations of DNA contamination in plasticware and water assumed to be sterile, which is not detected by standard DNA procedures. One technique commonly used to eliminate the presence of DNA is ultraviolet (UV) irradiation; we optimized such a protocol used in the treatment of water, tubes, plates, and tips for low copy number DNA (LCN) amplification. UV light from a Stratalinker® 2400 was administered to 0.2, 1.5 mL tubes, and PCR plates contaminated with up to 500 pg of DNA. They were subsequently quantified with an ALU‐based real‐time PCR method using the Rotorgene 3000. Overall, there was a decrease in concentration of DNA recovered as the duration of treatment increased. Nonetheless, following 45 min of irradiating a PCR plate with 500 pg of DNA, nearly 6 pg were still detected. However, when the plate was raised within an inch of the UV source, less than 0.2 pg of DNA was detected. Additionally, lining the area around the samples with aluminum foil further reduced the amount of time necessary for irradiation, as only 30 min eliminated the presence DNA in the raised PCR plate. Similar experiments were conducted using tubes filled with a solution of DNA and water in equivalent concentrations for 50, 15, and 1.5 mL tubes with comparative results. It is plausible that the aluminum foil increased the amount of reflection in the area thereby enhancing penetration of UV rays through the walls of the plasticware. This protocol was tested for the possibility of inhibitors produced from irradiation of plastic tubes. As our protocols require less irradiation time than previous studies, PCR sensitivity was not affected. Moreover, the lifespan of the UV lamps was extended. Our findings demonstrate that this method is useful as an additional precautionary measure to prevent amplification of extraneous DNA from plasticware and water without compromising the sensitivity of LCN DNA amplifications.

Prediction of eye and skin color in diverse populations using seven SNPs.

An essential component in identifying human remains is the documentation of the decedents visible characteristics, such as eye, hair and skin color. However, if a decedent is decomposed or only skeletal remains are found, this critical, visibly identifying information is lost. It would be beneficial to use genetic information to reveal these visible characteristics. In this study, seven single nucleotide polymorphisms (SNPs), located in and nearby genes known for their important role in pigmentation, were validated on 554 samples, donated from non-related individuals of various populations. Six SNPs were used in predicting the eye color of an individual, and all seven were used to describe the skin coloration. The outcome revealed that these markers can be applied to all populations with very low error rates. However, the call-rate to determine the skin coloration varied between populations, demonstrating its complexity. Overall, these results prove the importance of these seven SNPs for potential forensic tests.

Verification of eye and skin color predictors in various populations

Validation of testing methods is an essential feature in all scientific endeavors, but it is particularly important in forensics. Due to the sensitive nature of these investigations and the limited sample size it is crucial to validate all employed procedures. This includes novel forensic phenotypic DNA tests, to learn more of their capabilities and limitations before incorporating them as routine methods. Ideally, validations are performed on large sample sets that mimic real cases. Recently, three phenotypic predictors, two for eye colors and one for skin color have been published (Spichenok et al., 2011; Walsh et al., 2011). These predictors are well-defined by a selection of single nucleotide polymorphisms (SNPs) and unambiguous instructions on how to interpret the genotypes. These standardized approaches have the advantages that they can be applied in diverse laboratories leading to the same outcome and offer the opportunity for validation. For these tests to be used on the characterization of human remains, they should be validated on various populations to perform reliably without prior knowledge of ethnic origin. Here, in this study, these eye and skin color predictors were validated on new sample sets and it could be confirmed that they can be applied in various populations, including African-American, South Asian (dark), East Asian (light), European, and mixed populations. The outputs were either predictive or inconclusive. Predictions were then compared against the actual eye and skin colors of the tested individuals. The error-rates varied; they were low for the predictors that describe the eye and skin color exclusively (non-brown or non-blue and non-white or non-dark, respectively) and higher for the predictor that describes individual eye colors (blue, brown, and intermediate/green), because of uncertainties with the green eye color prediction. Our investigation deepens the insight for these predictors and adds new information.

Estimating the number of contributors to two-, three-, and four-person mixtures containing DNA in high template and low template amounts

Aim To develop guidelines to estimate the number of contributors to two-, three-, and four-person mixtures containing either high template DNA (HT-DNA) or low template DNA (LT-DNA) amounts. Methods Seven hundred and twenty-eight purposeful two-, three-, and four-person mixtures composed of 85 individuals of various ethnicities with template amounts ranging from 10 to 500 pg were examined. The number of alleles labeled at each locus and the number of labeled different and repeating alleles at each locus as well over all loci for 2 HT-DNA or 3 LT-DNA replicates were determined. Guidelines based on these data were then evaluated with 117 mixtures generated from items handled by known individuals. Results The number of different alleles over all loci and replicates was used to initially categorize mixtures. Ranges were established based on the averages plus and minus 2 standard deviations, and to encompass all observations, the maximum and the minimum values. To differentiate samples that could be classified in more than one grouping, the number of loci with 4 or more repeating or different alleles, which were specific to three- and four-person mixtures, were verified. Misclassified samples showed an extraordinary amount of allele sharing or stutter. Conclusions These guidelines proved to be useful tools to distinguish low template and high template two-, three-, and four-person mixtures. Due to the inherent higher probability of allele sharing, four-person mixtures were more challenging. Because of allelic drop-out, this was also the case for samples with very low amounts of template DNA or extreme mixture ratios.

Comment on “Low copy number typing has yet to achieve “general acceptance”” by Budowle, B., et al., 2009. Forensic Sci. Int. Genetics: Supplement Series 2, 551–552

Bruce Budowle, Arthur Eisenberg, and Angela van Daal recently authored an article entitled ‘‘Low Copy Number typing has yet to achieve ‘general acceptance’’’ [1]. We have concerns about both the title and the content of this article. First, the ‘general acceptance’ reference in the title of this article is not a scientific conclusion; rather it is a legal standard concerning the admissibility of scientific evidence as determined by a court of law. Whether a scientific method has ‘achieved’ general acceptance within the relevant scientific community is a determination reserved for the Court. Moreover, with a reference list limited to testimony given by one of the authors in a single Frye hearing in New York City and two of the authors’ own publications, we do not think the authors provide a scientific basis for their conclusions. It should be noted that the criminal case referenced in this article, People v. Megnath, is an open, ongoing homicide prosecution, with trial scheduled to commence in early 2010. We believe it is not a generally accepted practice within the forensic community to publish opinions on the results of testing where the criminal case is not yet concluded; however, we have prepared this response because we feel the substance of the authors’ statements merits a timely reply. Most of the authors’ assertions regarding the Office of Chief Medical Examiner [‘‘OCME’’] Forensic Biology Laboratory, particularly those wherein Dr. Budowle’s own testimony is referenced [2] are inaccurate or are taken out of context and misrepresent OCME’s protocols and practices. We clarify these misinterpretations below, specifically those surrounding six statements in the article upon which the authors base their opinions. The first of these statements reads as follows: ‘‘stochastic effects and potential allele drop out have to be considered for RFU values as high as 2000 RFUs.’’ The authors interpret this statement to mean a stochastic threshold of 2000 RFUs should be used. However, validation studies of Low Template DNA [LT-DNA] testing performed by the OCME, demonstrated that a stochastic threshold based on peak heights was not a reliable indicator of allelic dropout. Due to the enhancement of our amplification system and injection parameters for capillary electrophoresis, peak heights above 2000 RFUs were observed for samples amplified with as little as 6 pg of DNA. Despite that the peak

Interpretation Guidelines for Multilocus STR Forensic Profiles from Low Template DNA Samples

Low template (LT) DNA testing is a more sensitive method of PCR DNA typing which tests lower quantities of DNA compared to traditional PCR DNA protocols. Methods applied in this testing involve amplification or postamplification efforts to increase detection sensitivity. Establishing the interpretation rules of the results obtained is condition sine qua non for successful incorporation of this valuable technique into forensic casework. Here we describe a successfully optimized and validated approach to interpretation of LT-DNA samples.