Advantages of the Casework Direct System for Processing Touch DNA Samples
Daniela de Oliveira Francisco, FMUSP, Fernanda de Todelo Gonçalves, University of São Paulo, Cintia Fridman, University of São Paulo, and Luis Fernandez Lopez, University of São Paulo
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In a forensic routine, the methods used to process samples obtained from a crime scene are crucial to the success of all operations performed at the laboratory. Inappropriate collection, extraction, or amplification methods can compromise an investigation. Since many DNA extraction methods currently utilized do not recover all of the DNA collected from the crime scene, studies have focused on optimizing these techniques.
For touch DNA samples, these improvements are extremely important because this type of sample has a low concentration of DNA, which can create challenges in downstream analyses. New methodologies for touch DNA samples are needed to optimize extraction by removing inhibitors, while also avoiding DNA loss as you move from one step to another in the workflow.
The DNA IQ™ System (DNA IQ) is an extraction kit based on magnetic beads for purification of nucleic acids. This kit was developed for forensic purposes, to enable the user to extract DNA from a variety of sample types, including stains and liquid samples. The process includes multiple wash steps to remove PCR inhibitors and contaminants frequently encountered in casework samples. Due these advantages, DNA IQ is the current method used in many laboratories around the world, including for forensic analysis in São Paulo city, Brazil.
Recently, the Casework Direct System (Casework Direct) was developed by Promega as a new method of extraction based on a no-wash protocol, maximizing the chance of recovering DNA collected from casework samples. Studies have shown that the Casework Direct System is a suitable, accurate, and reproducible method for the rapid isolation of DNA, offering the possibility to replace other techniques when used to process several challenging forensic sample types. To test its potential for use with touch DNA samples, we conducted comparative studies, considering variables that can interfere with DNA deposition or collection, such as the type of surface and environmental factors.
Fifty-two simulated crime samples were done on computer keyboards, three types of knife handles (plastic, wood, and steel), exterior door handles and automobile steering wheels. Eight of these 104 experiments were performed on computer keyboards, 48 on the various knife handles, and 48 on exterior door handles of cars or their respective steering wheels (Figure 1).
Fig. 1. Scheme of experiments performed on: (a) computer keyboards; (b) knife handles; and (c) steering wheels and exterior door handles of cars. Each line refers to a single experiment.
Each experiment was conducted with two individuals (first and second donors) aiming to obtain a mixture profile of the two individuals. Experiments on exterior door handles were performed under different environmental conditions: sunny/hot days and rainy/cold days.
The biological material deposited by these individuals on each tested surface was collected by the double swab method 5 minutes after deposition, and decontamination of surfaces was performed immediately after each experiment with 70% alcohol. To compare two different methodologies of extraction (DNA IQ and Casework Direct System), all the experiments were done in duplicate, i.e., half of the experiments obtained from a surface were extracted by one method and the remaining half was extracted by the other.
After the extraction, all samples were quantified with Quantifiler Trio™ (Applied Biosystems) and amplified with the PowerPlex® Fusion System (Promega Corporation). Parameters of cycling were adjusted for 31 cycles and the amplified products were injected for capillary electrophoresis on the ABI3130 Genetic Analyzer (Applied Biosystems). The profiling analysis was conducted by GeneMapper® ID software, version 3.2.
Reference samples: Using FTA® cards (Whatman), blood samples were collected from all participants in order to generate reference profiles that were used for a comparison with the mixture profiles obtained from the experiments.
From 104 samples collected, it was possible to generate 83 STR profiles (79.8%) in total, with 32 out of 52 from the samples processed with DNA IQ (61.5%), and 51 out of 52 from the samples processed with the Casework Direct System (98.1%). The remaining 21 samples (20.2%) did not produce any profile. Experiments that failed are summarized in Table 1.
Since the allelic composition of each individual that comprises the mixture was known, the total number of STR markers with allelic correspondence between the observed and the expected profile was counted and represented as a percentage. Table 2 shows the average percentage of alleles recovered from each participant for samples extracted with DNA IQ and Casework Direct, respectively.
The utility of these profiles was evaluated according to the Operational Procedures Manual of the National DNA Index System (NDIS), which established that at least 8 STR markers present in the original CODIS Core Loci are necessary to consider a STR profile useful to an investigation. Therefore, out of the 83 profiles obtained, only those with at least 8 STR markers from the second donor in the mixture (presenting partial or full genotypes) were considered. Thirty-one of the 83 STR profiles (37.3%) would be useful for this purpose, with 24 (77.4%) from the Casework Direct-processed samples and 7 (22.6%) from the DNA IQ-processed samples.
Since 2017, seven additional markers have been included in the CODIS core, for a total of 20 STR markers. Accordingly, we did another evaluation considering at least 8 STR markers corresponding to the second donor in the mixture for any of these 20 STR (partial or full genotypes). In this case, 42 of the 83 STR profiles (50.6%) would be useful, with 30 (71.4%) from the Casework Direct-processed samples and 12 (28.6%) from the DNA IQ-processed samples.
Table 3 shows the results obtained from the samples processed with the respective method and surface type as for Table 2.
A Kruskal-Wallis test demonstrated that surface type significantly affects recovery when using either extraction method (P = 0.005 for DNA IQ and P = 0.002 for Casework Direct). The box plot in Figure 2 shows that the surface type affects the kits’ performance in a similar way, but that Casework Direct performs slightly better in general.
A chi-square test showed that Casework Direct produced significantly more useful profiles than DNA IQ (P =0 .001), based on NDIS analysis.
Our results showed that the Casework Direct System is better for recovering DNA from touch DNA sources, since only one sample processed with this method failed, while 20 samples extracted with DNA IQ yielded no usable STR profile. Apparently, the Casework Direct System increases the chance of generating usable DNA profiles even if the surface is not optimal for this type of analysis, as in the case with hard, polished, and low-porous surfaces, such as a steel knife handle. Additionally, samples extracted with the Casework Direct System resulted in superior STR profiles when compared to samples extracted with DNA IQ, making it more effective to identify both contributors in the mixture.
The higher efficiency demonstrated by the Casework Direct System could be explained by the differences found in the protocols of these two extraction methods. The DNA IQ System is based on successive washes of a paramagnetic resin to capture a consistent amount of DNA. After DNA capture by magnetic particles, successive washes occur to purify the DNA prior to the amplification process. However, studies reported that, from one wash to another, a portion of the genetic material can be lost because not all of the DNA binds to the resin. Considering that touch DNA samples already have low DNA amount in their composition, any loss due to a wash could explain the inability to obtain a useful STR profile—or any STR profile at all—from a process that has wash steps. The Casework Direct System has no wash step, and contains a buffer and a reducing agent capable of producing a DNA lysate from casework samples in approximately 35 minutes, minimizing DNA loss.
Differences observed in the quality of profiles obtained from the different objects used in our study also could be explained by the various features of the surfaces, which often influence the transfer, adhesion, and recovery of the trace deposited. It is already known that porous surfaces retain more cells than non-porous surfaces, which have greater adhesion capacity. As observed, steering wheels were the best surface from which to recover DNA and generate usable profiles, while knife handles were the worst surfaces from which to do so.
Results obtained from Kruskal-Wallis test analysis demonstrated that the surface type significantly affects DNA recovery when using either extraction method. Both kits perform differently depending on the surface (P = 0.005 for DNA IQ and P = 0.002 for Casework Direct), but the Casework Direct System samples were better in general. This new extraction kit could be, therefore, a more efficient method to help solve crimes where DNA has likely been deposited on surfaces like those tested, as suggested by the chi-square test results which showed a significant difference between both kits to obtain a useful profile (P = 0.001).
Surprisingly, even for exterior door handles of cars exposed to rain, the Casework Direct System method was a good alternative for extraction. Our results showed that 62.5% of the profiles would be useful for forensic purposes, despite the knowledge that environmental factors, such as high exposure to light, high temperature, or moisture can degrade DNA and impair its analysis.
This study provides insights about the efficiency of Casework Direct System for touch DNA samples analysis on different surfaces and varying environmental conditions.
Compared with DNA IQ System, the Casework Direct System seemed to be more promising for these purposes, showing a higher number of alleles recovered from both donors in the mixtures for almost all the surfaces tested, enhancing the chance of identifying an individual. However, although the possibility of generating STR profiles from DNA deposited on computer keyboards, knife handles, or steering wheels and door handles of cars could be demonstrated, in many cases the profiles generated would not be useful to identify the person of interest. This is especially true, for those from by non-porous surfaces, like steel handle knives, because of the insufficient number of STR markers amplified.
Despite the challenging nature of touch DNA samples, the collection of DNA should not be overlooked in any criminal case, since new methods are being developed to help overcome the inherent challenges with this sample type.
Daniela de Oliveira Francisco
Daniela de Oliveira Francisco graduated in Biology at Universidade Presbiteriana Mackenzie (UPM) and later interned at the Faculdade de Medicina da Universidade de São Paulo (FMUSP), researching polymorphisms related to endometriosis. Currently, she is a Doctorate student at FMUSP, developing her project at the Molecular Biology and Forensic Genetics laboratory, focused on Touch DNA study to assist the work of Brazilian forensic expertises.
Fernanda de Todelo Gonçalves
Fernanda de Todelo Gonçalves graduated in Biological Sciences from UNESP in São José do Rio Preto. She holds a PhD in Sciences from the Pathology program of the Faculty of Medicine of the University of São Paulo (USP) and is currently a scientific researcher at the Laboratory of Immunohematology and Forensic Hematology of the Faculty of Medicine of the University of São Paulo. Has experience in Genetics, with an emphasis on Human and Medical Genetics and Forensic Sciences, acting mainly on the following themes: Molecular Epidemiology of Head and Neck Cancer, Malignant Cutaneous Melanoma, Xenobiotic and Alcohol Metabolization Gene Polymorphisms, Human DNA Identification, Paternity Test, Forensic Phenotyping and Touch DNA.
Cintia Fridman is graduated in Biological Sciences (1991), Master in Biology/Genetics (1995), PhD in Biology/Genetics (1999) and Post Doctorate by the Biosciences Institute (2001) in São Paulo University (USP), working with Prader-Willi and Angelman Syndrome. She has a Post-Doctorate at the Institute of Psychiatry-of USP (2003) working with genetics of psychiatric diseases. She is currently a professor at the University of São Paulo, in the Department of Legal Medicine. She is responsible for the LIM40-HCFMUSP Genetics and Molecular Biology laboratory. She experience in Genetics, with an emphasis on Forensic Genetics and Human and Medical Genetics, acting mainly on the following topics: human identification, forensic genetics, mitochondrial DNA, SNP, genetic ancestry, forensic DNA phenotyping, STR, Y chromosome, polymorphisms.
Luis Fernandez Lopez
Luis Fernandez Lopez is currently Professor of Mathematical Epidemiology and Medical Informatics in the Department of Forensic Medicine, Faculty of Medicine, University of São Paulo and professor at the Center for Internet Augmented Research and Assessment at Florida International University, in Miami, USA. He is also Principal Investigator of the ANSP project (an Academic Network at São Paulo), funded by FAPESP (São Paulo Research Foundation) and co-Principal Investigator of the AmLight project (Americas Light paths), co-financed by FAPESP, RNP (National Teaching and Research Network) and NSF (National Science Foundation in USA).