The National Institute of Justice (NIJ), in conjunction with the National Forensic Science Technology Center (NFSTC), is sponsoring this workshop which is designed to help facilitate the transition of novel technologies into practice by operational forensic facilities. This Technology Transition Workshop, which highlights technologies developed under NIJ’s forensic science research and development programs, is a critical component of NIJ’s research, development, testing and evaluation (RDT&E) efforts.
This Rapid Biological Screening & Analysis Methodologies for Improving Throughput Technology Transition Workshop focuses on research projects that have the capability to increase the throughput of biological samples in one of two ways: through a screening process that serves to enable selection of critical samples requiring a more complete analysis either at the scene or in the laboratory, or via a fully integrated rapid DNA analysis platform system.
Using the website created for this workshop, attendees will be required to review provided introductory microfluidics lectures and selected journal articles prior to the first installment of this workshop on October 24, 2011. The daily meetings of the workshop will be delivered via approved-access WebEx training sessions on the Internet. Each day of the workshop, a live question-and-answer period with the session’s instructor or instructors will be held in real time immediately following broadcast of the pre-recorded lecture(s).
Rapid Human Forensic Identification: The ANDE System and Components
Richard Selden, M.D., Ph.D.
Dr. Selden will provide attendees with an understanding of NetBio’s system for Rapid DNA Analysis (RDA)—the fully integrated ANDE (Accelerated Nuclear DNA Equipment) system. To use ANDE, the operator places buccal swabs into a biochipset, inserts the biochipset into the instrument, and presses start—no other interventions are required. The reagents are factory-loaded into the biochipsets, further increasing the ease of use of the system. The technical details of each element of the ANDE system will be discussed, along with the integration of these elements: sample preparation and DNA purification from buccal swabs, rapid thermal cycling, highly multiplexed amplification, and optical detection and sizing of DNA fragments. The specifics of the system design, which allows for automated microfluidic workflows that enable its use by a non-technical operator in the laboratory and in the field, will be highlighted. Finally, Dr. Selden will discuss current work to automate processing of casework samples and touch samples. Electropherograms generated from analysis of samples will be available for review.
Time Since Deposition (TSD) of Dried Bloodstains Using Ultraviolet/Visible (UV-VIS) Spectrometric Analysis of Hemoglobin
Jack Ballantyne, Ph.D.
The potential to evaluate the time since deposition (TSD) of a bloodstain found at a crime scene will be discussed during this lecture by Dr. Ballantyne. A historical overview of various methods of establishing TSD, along with the challenges of these approaches, will be presented. During the lecture, Dr. Ballantyne will outline the UV-VIS spectrophotometric analysis method developed by the University of Central Florida for the estimation of TSD of dried bloodstains, which is based on hemoglobin’s characteristic oxidation chemistry. Research results demonstrating the ability to make a distinction between bloodstains that were deposited minutes, hours, days and weeks prior to recovery and analysis will be presented. The sensitivity of the developed testing method will also be discussed. Further, the option for evaluating the potential value of bloodstains in situ to assist in determining their probative value and need for collection will be presented, given the possibility of performing TSD measurements at the crime scene using a portable low-sample-volume spectrophotometer.
Identification of Fetal Blood Using Developmentally Regulated Gamma Hemoglobin mRNA Isoforms
Jack Ballantyne, Ph.D.
Research results indicating the potential to evaluate bloodstains to determine the age category of the depositor will be presented. During his lecture regarding the identification of developmentally regulated gamma hemoglobin messenger RNA (mRNA) isoforms, Dr. Ballantyne will illustrate the ability to use this technique to definitively determine that a bloodstain has originated from a newborn. The multiplex quantitative reverse transcription PCR (qRTPCR) assays incorporating the identified novel newborn-associated mRNAs that have been designed, tested, and evaluated for their potential forensic use will be presented. The molecular characterization and tissue expression of these identified gamma hemoglobin isoforms will also be discussed. Additional research depicting the potential ability to assign the age of the individual who deposited a bloodstain into one of four age groups—newborn, adolescent, middle-aged, or elderly—based on an assessment of the mRNA age-specific gene expression patterns will be addressed.
Rapid Melt-Based STR Prescreening of Forensic Samples at the Crime Scene
Micah Halpern, M.S.
Mr. Halpern will provide the attendees with an introduction to DNA melt curve analysis with a focus on its use as a unique alternative method for short tandem repeat (STR) genotyping. The benefits of utilizing a melt-based approach versus standard methods will be addressed, as will details of the research accomplished to date for chemistry development, microfluidic-based assay incorporation, and forensic validation. Details will also be provided on the current stage of the chemistry/platform and potential impact on the practice of forensic science.
A Real-Time Multiplex SNP Melting Assay to Discriminate Individuals
Eric Buel, Ph.D.
During this lecture, Dr. Buel will describe a method that quickly and inexpensively differentiates crime scene samples from multiple donors which is available for use in expediting casework analysis by easily allowing the selection of probative items requiring comprehensive testing. Information regarding the development of multiplex single nucleotide polymorphism (SNP), fluorescence resonance energy transfer-based real-time PCR assays to fill this need will be presented. The two alternate 6-plex assays (with and without gender determination) that have been developed for the six-color RG6000 real-time instrument (Corbett Robotics, Inc.) and one seven SNP plus gender assay (performed as two 4-plex assays, one with gender, the other without) that have been developed for use in four-/five-color real-time instruments will also be discussed. Another approach to screening samples using melt-based analysis will also be presented. This method allows for simpler “kit” preparation for sample analysis than the SNP-based method and may be easier to incorporate into normal analytical schemes.
At the completion of the Rapid Biological Screening & Analysis Methodologies for Improving Throughput Technology Transition Workshop, the participant will have acquired the knowledge to:
- Discuss microfluidic elements and functions, the history of microfluidics, and the application of microfluidics to HID analyses
- Describe the ANDE Rapid DNA Analysis platform and its applications in the laboratory and field environments
- Describe the operation and performance of the ANDE system and the role of the forensic laboratory in evaluating the system and optimizing its utility
- Describe the analytical process available for use in determining the TSD of a bloodstain and explain the limitations of this and other analysis options for this purpose
- Explain how the process for assessing the mRNA age-specific gene expression patterns works
- Describe the process being developed for determination of the physical age of an individual who deposited a bloodstain, including the current assays for determining that a bloodstain originated from a newborn
- List the considerations and limitations for STR analysis by traditional capillary electrophoresis methods
- Describe the DNA melt curve analysis and analytical approaches, as well as the unique STR melt genotyping approach
- Discuss the research results for STR melt genotyping chemistry, microfluidic assay compatibility and preliminary forensic validation efforts and impacts for forensic analysis
- Explain the multiplex SNP fluorescence resonance energy transfer-based real-time PCR assays available to quickly and inexpensively differentiate crime scene samples from multiple donors