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A Private and Public Sector Partnership for the analysis of Convicted Felon Database Samples and Non-suspect Casework.
Teresa H. Aulinskas1, Ph.D., George R. Riley1 Ph.D., Piper L. Schwenke1, B.S.,
Amy L. Palm, B.A.1, Katie Woodard1, B.S., Jennifer L. Iem1, M.S., Anne G. Pace1,
B.S., Todd Bille2, M.S., Paul Misner2, Eric Lawrence2, and Howard C. Coleman1,
B.S., B.A,
INTRODUCTION Genelex Corporation and the Indiana State Police Crime Laboratory (ISP) have formed a partnership to build a database of convicted felon DNA profiles and to analyze non-suspect casework evidence. Both of these programs use the thirteen core CODIS STR (short tandem repeat) loci analyzed using Promega PowerPlex technology. CODIS (Combined DNA Index System), the FBI-developed software system for analyzing and tracking DNA profiles, has been installed in 94 laboratories in 41 states. As of June 1998, CODIS had generated more than 400 matches between database samples and crime scene evidence. These "blind" or "cold" hits have typically led to the resolution of serious, difficult cases. The number of convicted felon samples requiring DNA profiling has exploded. As of June 1998, all 50 states had passed legislation requiring offenders convicted of a variety of crimes to provide samples for DNA analysis. Of the greater than 600,000 samples collected less than half have been analyzed. This backlog is exacerbated by advances in DNA analysis methods that generate data incompatibilities.
IMPACT OF DNA ANALYSIS ADVANCES ON FELON DATABANKS. Over the past decade RFLP (restriction fragment length polymorphism), has been the predominant method used to analyze both offender database samples and criminal casework. With the advent of PCR (polymerase chain reaction) and its many advantages, DNA profiles incompatible with RFLP have been generated. These include DQA1, Polymarker and more recently the STR loci that are coming into common usage. The implementation of STRs represent the third major wave of forensic DNA analysis methods and can be expected to survive as the predominant method for a relatively long period of time. STRs combine the speed and sensitivity of PCR with a level of discrimination approaching that of RFLP. The FBI has designated thirteen STR loci as the core set that must be included in convicted felon DNA profiles that are accepted into NDIS (National DNA Index System). The overall result of these changes in methodology are overwhelmingly positive but they have delayed the input of DNA profiles into CODIS and subsequently into NDIS. The move from RFLP to STRs has been time consuming and expensive, as it has engendered a new round of training, validation and implementation at federal, state and local levels. This evolution in technology has resulted in many laboratories seeing their RFLP databases become obsolete as they convert to PCR-based methods. In such cases, no matches can be made between database samples and crime scene evidence. STR analysis has been further complicated by a choice of reagent kits and equipment that produce compatible data, but do not perform the analysis in precisely the same fashion. DATABASE SAMPLE ANALYSIS The essential elements of the DNA analysis partnership between the Indiana State Police Crime Laboratory and Genelex are outlined in the flowchart.
The workflow starts with the documentation, collection, and shipping of felon samples by the ISP Crime Laboratory. High-throughput sample processing, DNA analysis and CODIS formatting of data occur at Genelex. The data and samples are then delivered back to the ISP Crime Laboratory for input into CODIS and NDIS. Several aspects of the database work are common to both laboratories. These include validation standards and QA & QC requirements including accreditation. Accreditation by ASCLD/LAB is preferred but not required. Both laboratories, however, must follow federal DAB (DNA Advisory Board) standards if the data is to be acceptable to NDIS. PARTNERSHIP BETWEEN INDIANA STATE POLICE AND GENELEX CORPORATION The Indiana State Police and Genelex Corporation have been working together using this team approach to database analysis since 1997. The essential elements of this partnership are presented in the following flowchart. Both laboratories validated and implemented the Promega PowerPlex 1 STR system for the Hitachi FMBIO in 1997. Using this system more than 18,000 samples have been analyzed by Genelex. This has been followed by the validation and implementation of PowerPlex 2. To date we have run over 3,500 samples with PowerPlex 2.
Fig 1. PowrePlex 1. DNA was extracted from punched 903 Paper or FTA paper (FTA protocol, Life Technologies). Amplification using the GenePrint PowerPlex 1.2 identity kit (Promega Corporation) was as recommended by the manufacturer's protocol for the Perkin Elmer 480 thermal cycler. Amplified samples were separated by PAGE on 43 cm gels (SA-32, Life Technologies Inc.) using 4.5% acrylamide:bisacrylamide (19:1), 1xTBE, 7M urea. Electrophoresis was at 65 W for 1.5 hr (pre-run ~0.3 hr at 65W). Bands were detected by scanning with an FMBIO II (Hitachi) and were analyzed with Hitachi Analysis 6.0 and STARcall software. STR ANALYSIS USING POWERPLEX KITS AND THE HITACHI FMBIO STR analysis is routinely performed using the Geneprint PowerPlex 1 and 2 Fluorescent STR systems from Promega Corporation, and the Hitachi FMBIO II fluorescent scanner. Each PowerPlex system allows the simultaneous amplification and single lane detection of eight or nine STR loci. The fluorescent scanner performs automated three color detection of the STR loci and the internal lane marker. PowerPlex 1 allows the simultaneous amplification of eight STR loci. Depicted in Figure One is one of the laser-activated fluorescent channels, showing the separation of four DNA loci. Each lane represents the DNA profile of a single offender.
Fig 1. PowrePlex 2. DNA profiles were developed as described in Fig 1. Amplification using the GenePrint PowerPlex 2.1 identity kit (Promega Corporation) was as recommended by the manufacturer's protocol for the Perkin Elmer 480 thermal cycler. PowerPlex 2 allows the simultaneous amplification of eight STR loci and Amelogenin, the gender typing locus. Depicted in Fig 2. is the one of the three channels, showing the separation of five STR loci. Three of the STR loci in the PowerPlex 2 system, TPOX, vWA and THO1, are also present in PowerPlex 1, providing an excellent check for quality. DATABASE WORK FLOW Efficient and effective processing of samples requires workflow streamlining. At Genelex, samples are accessioned into a computerized laboratory information system. Samples are batched by computer and forms and tube labels generated. DNA extraction is simplified. Samples on 903 paper are punched and processed by washing with FTA reagent, thereby eliminating DNA quantitation. Multiplex amplification of STRs is followed by PAGE (polyacrylamide gel electrophoresis) using sharks tooth combs for maximum sample loading per gel. Extender plates (43 cm) and an external aluminum heat sink are used for maximum resolution and edge-to-edge linearity. Fluorescent analysis is followed by analytical review, rejection of problematic samples, and calling of STR alleles. The use of internal lane markers greatly reduces the occurrence and interpretation of migration anomalies. CODIS files are generated and a final technical review preformed. Electronic and paper data are delivered to the Indiana State Police Crime Laboratory where data review, quality checks and input of the DNA profiles into CODIS takes place.
A computerized laboratory information system is essential for tracking and links have been made between the laboratory information system, the Hitachi FMBIO II and the CODIS programs. Efficient, effective DNA extraction is also critical. We have observed high re-run rates with chelex extraction of blood stains on 903 paper. Re-run rates have been low with the FTA wash procedure and PowerPlex 1. Unfortunately re-run rates are higher with PowerPlex 2. As with all high-throughput operations, technical problems can lead to large batch failures due to the rapidity of processing. In our experience, results requiring communication between facilities, such as stutter bands, low intensity bands and locus drop-out have been minimal. DATABASE STAFF The Indiana State Police Crime Laboratory team consists of a database supervisor, a technical leader, and analysts performing quality assurance and administrative tasks. Genelex's team consists of a laboratory director, technical leader, several analysts and an administrator. Both laboratories have approximately 2.7 people (full time equivalent) working on this project. This translates to about 200 samples, profiled by the 13 core loci, per person, per month. NON-SUSPECT CASEWORK The Indiana State Police Crime Laboratory has recently arranged for Genelex to process some of their non-suspect forensic casework evidence. The DNA Identification Act of 1994 authorized the FBI to establish DNA indexes for: 1) persons convicted of crimes; 2) samples recovered from crime scenes; and 3) samples recovered from unidentified human remains. We have recently begun processing samples recovered from old and new crime scenes in which there is no suspect. The Indiana State Police Crime Laboratory estimates that they will process approximately 600 non-suspect cases per year. It is estimated that casework will fall into the catagories outlined in the following graphic.
The Indiana State Police Crime Laboratory Crime Laboratory will screen evidence and only those cases that can be expected to have a reasonable amount of DNA are sent to Genelex. It is expected that approximately 50% of rape cases will be negative for semen. Approximately 300 cases per year are expected to require DNA analysis. Of those cases, the ISP Crime Laboratory will process rape evidence samples which exhibit low sperm count. Evidence samples exhibiting high sperm count will be sent to Genelex for DNA typing. NON-SUSPECT CASE WORK FLOW Non-suspect casework procedures take advantage of the testing efficiencies developed and implemented in high throughput convicted felon testing. Each of the steps from extraction through analysis and reporting has been streamlined to reduce both the amount of processing time and the amount of paperwork required. Casework flow between the two labs in represented in the following graphic.
Non-suspect sample processing starts at the Indiana State Police Crime Laboratory where analysts screen the evidence and perform body fluid ID testing. The ISP Crime Laboratory retains a portion of all samples including those sent to Genelex. A simplified chain of custody between the two laboratories has been developed. At Genelex, several cases are batched and twelve to fifteen samples processed simultaneously. DNA extraction is more complicated when compared to database analysis. Rape evidence swabs are processed by differential organic extraction. STR analysis is also more complicated as mixtures of DNA from more than one individual are observed in some cases.
Figure 3. Non-suspect casework analyzed by PowerPlex 1. Purified human DNA was obtained from questioned evidence by organic extraction (phenol-chloroform) and from known reference samples on FTA paper (FTA protocol, Life Technologies). Quantitation of human DNA was by slot blot using radiolabeled probe to the locus D17Z1. Amplification, electrophoresis and analysis is as described in Figure 1. Three rape and two burglary cases are shown. N = E1 or non-sperm cell fraction; S = E2 or sperm cell fraction; V = victim reference standard. DNA loci shown are D16S539, D7S820, D1S317 and D5S818). Figure 3 shows PowerPlex 1 data from several rape and burglary cases. Vaginal swabs were separated into non-sperm (N, E1 fraction) and sperm fractions (S, E2 fraction). As expected, the victim's sample matches the non-sperm epithelial cell fraction of the rape evidence swab. Some cases are complicated by the observation of mixtures. The non-sperm cell fraction in case three is a mixture, indicating incomplete separation of sperm. For non-suspect casework, gel electrophoresis follows the classic "forensic casework" gel set-up, with each sample flanked by the allelic ladder. Wider gel combs are also used to allow clear separation between samples. Analysis is followed by technical review; generation of CODIS output files and a custom report for each case. The Indiana State Police Crime Laboratory performs reviews and places the DNA profiles into CODIS. While the implementation of forensic casework and database analysis must meet comparable DAB requirements, the actual testing work-flow is markedly different between the two applications. When both systems are used together they address backlog in both CODIS databank samples and non-suspect cases. When implemented as a partnership between the public and private sectors, effectiveness can be optimized. A partnership increases both laboratory's ability to provide reliable, timely and economical data to the justice system. REFERENCES 1. Validation of PowerPlex STR multiplex and Amelogenin sex identification typing kits using the FMBIO II fluorescent scanner: Forensic casework and high throughput convicted offender databanking. George R. Riley, Ph.D, et al. In: Proceedings from the Eighth International Symposium on Human Identification. 1997, Promega Corporation pp. 53 - 55. 2. Automated Fluorescent Detection of STR Multiplexes - Development of the GenePrint PowerPlex and FFFL Multiplexes for Forensic and Paternity Applications. Schumm, James W,. et al. In: Proceedings from the Seventh International Symposium on Human Identification. 1996, Promega Corporation pp. 70-80. 3. Automated Fluorescent Detection of 8-locus and 4-locus STR Multiplexes. Schumm, James W,. et al. In: Proceedings from the Eighth International Symposium on Human Identification. 1997, Promega Corporation pp. 78-81. 4. Promega Corporation, GenePrint STR System Technical Manuals, # TMD008, TMD011.
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