In the United States, there is a substantial backlog of samples
requiring short tandem repeat (STR) DNA marker analysis. Approximately
200,000 to 300,000 collected convicted offender samples (U.S. Department
of Justice 2003) and more than 540,000 evidentiary samples where
there is no suspect (Attorney General's Report on the DNA Evidence
Backlog 2004) currently remain to be analyzed nationwide. Additionally,
500,000 to 1,000,000 authorized convicted offender samples have
not yet been collected (U.S. Department of Justice 2003).
Figure 1. Backlog of Convicted Offender Samples
To address this issue, Public Law 106-546, DNA Analysis Backlog
Elimination Act of 2000, was enacted on December 21, 2000.
This law authorized $170,000,000 toward reducing the backlog (DNA
Analysis Backlog Elimination Act of 2000).
- $15,000,000 was appropriated in 2001 through 2003 for DNA analysis
of samples to be included in the Combined DNA Index System (CODIS),
the nationwide DNA database.
- $25,000,000 to $50,000,000 was appropriated in 2001 through
2004 for DNA analysis of samples from crime scenes and to increase
the capability of public laboratories to carry out DNA analyses.
The intent of this spending was to eliminate the backlog of samples
requiring STR DNA analysis by providing additional manpower and
instrumentation for use with existing nuclear DNA analysis technologies.
Although existing nuclear DNA analysis technologies are valid and
accurate, they are also labor-intensive and time-consuming. Introducing
automation into the process flow for analysis of forensic biological
samples would overcome the backlog problem and prevent its recurrence.
Identification of probative biological samples, the technical steps for typing
the 13 core CODIS STR loci, and the interpretation of STR-analytical results and
associated data quality review could all be automated. This would assist in achieving
process quality and reproducibility.
During 2000 in support of
the backlog reduction efforts, the FBI's Counterterrorism and
Forensic Science Research Unit designed a research and development
plan for automating the forensic analysis of biological evidence,
which was funded by Congress in 2001. The FBI automation initiative
is divided into three main areas—serology, the
STR-typing process, and online data interpretation and quality
The goals for the serology initiative are to develop methods for
the definitive identification of all forensically relevant biological
stains and to automate the execution of these methods. Currently,
biological samples deposited at crime scenes are identified by visual
inspection, chemical reactions, enzymatic reactions, and standard
Figure 2. Serological Examination of Forensic Evidence
Both a presumptive test and a confirmatory test are performed in
the process, and these tests are conducted sequentially, requiring
a new sample for each test (Ballantyne 2000). However, definitive
tests do not exist for each of the frequently encountered body fluids
(e.g., saliva or urine). Operational efficiency could be improved
if a system existed whereby a complete panel of body fluid identification
tests was performed simultaneously from a single sample (i.e., multiplexed
analysis), and the identification system was amenable to automation.
The FBI recently initiated a project to develop a multiplexed immunoassay
to identify forensically relevant body fluids. Novel antigens not
previously used in forensic analysis will be interrogated using
monoclonal antibodies, the most specific immunological reagents
The immunoassays will first be developed in a format suitable for
individual high-throughput automation on a robotic liquid handler.
Subsequently, the individual assays will be multiplexed and adapted
to an as yet undecided detection platform. One possibility is a
suspension array based on flow cytometry (Kellar and Iannone 2002;
Nolan and Mandy 2001).
A second project to identify body fluid under development by the
FBI is the probing of messenger RNA (mRNA) species that are selectively
expressed in cells that collectively comprise a particular body
fluid (Juusola and Ballantyne 2003). Each cell type in the human
body has a unique pattern of gene expression that is manifested
by the presence and relative abundance of specific mRNA species,
the molecular intermediate between genic DNA, and expressed protein
(Caron et al. 2001).
Figure 3. Visual Inspection of Forensic Evidence
Studies have shown that
mRNA is stable in biological stains of forensic relevance and
can be recovered in sufficient quantity and quality for analysis
(Juusola and Ballantyne 2003). Candidate tissue-specific genes
have already been identified for saliva (statherin, histatin
3, PRB1, PRB2, and PRB3), but tissue-specific genes for other
body fluids of forensic importance have yet to be identified
(Juusola and Ballantyne 2003). When a panel of tissue-specific
genes has been identified for body fluids significant in forensic
investigations, a parallel analysis method will be developed
to probe these mRNA species. One possibility is a microarray
of complementary DNAs, or sequence-specific oligonucleotides,
capable of recognizing each of the candidate genes in a "chip" format
(Juusola and Ballantyne 2003).
Two parallel strategies are planned for automating the technical aspects of
forensic nuclear DNA analysis. First, some of the manual steps in the STR-typing
process will be replaced by automated systems performing these tasks or by new
procedures that are less labor-intensive. Second, a completely dedicated, automated
system for forensic genotyping will be developed. Improvements in the steps of
the STR-analysis procedure can be developed in the short-term and introduced into
casework within two to three years. Developing an integrated STR-analysis system
is a longer-term project and estimated to take between three and five years.
Automating Individual Steps in the STR-Typing Process
STR-analysis involves the following four steps:
- Extraction of DNA from biological samples
- Quantification of the obtained DNA
- Amplification of the CODIS-required STR loci by the polymerase
chain reaction (PCR)
- Separation and detection of the amplification products by capillary
The PCR amplification of template and subsequent production of
DNA patterns by capillary electrophoresis already are semiautomated,
as they involve little user intervention. Extraction and quantification
of DNA remain to be automated.
Figure 4. Set-up of the Polymerase Chain Reactions for
Amplifying the CODIS-Required STR Loci
Extraction and purification of nucleic acids from biological samples
is one of the most difficult and tedious tasks in forensic DNA typing.
DNA extractions from bloodstains, semen stains, buccal swabs, or
liquid blood samples entail separating DNA molecules from proteins
and other cellular material through a manual process.
For mixtures of epithelial and sperm cells, as in the case of vaginal
swabs, the cells must first be separated prior to extracting the
DNA. This is accomplished by simple chemistry. The swab is incubated
in a detergent/proteinase K solution to lyse the nonsperm cells.
Then the sperm nuclei are lysed by incubation in a detergent/proteinase
K/dithiothreitol solution and purification of the DNA follows from
each of the cell lysates. This procedure is known as differential
extraction (Gill et al. 1985).
One of the FBI's research projects in this area is to build an
instrument with a microfluidic platform that will perform differential
and nondifferential DNA extractions. The core of this system will
be an ultrasonic module for selective cell lysis and a silicon chip
module for rapid DNA extraction, purification, and concentration.
Another project involves evaluating several DNA extraction procedures
that are amenable to execution on a robotic liquid handler and programming
a liquid handler to perform some of those processes. At the same
time, a robotic liquid handler will be programmed to execute a proprietary
differential extraction procedure that is based on differential
lysis and selective filtration.
The detection and quantification of human nuclear DNA is another
labor- and time-intensive step in the forensic STR-analysis procedure.
The most popular method for quantifying human DNA in forensic laboratories
today involves hybridization with a primate-specific alpha satellite
DNA sequence located on chromosome 17, D17Z1 (Walsh et al. 1992).
Figure 5. Quantification of DNA by Hybridization to Human
In this procedure, a slot-blot apparatus is used to capture genomic
DNA and a serial dilution of a human DNA standard on a nylon membrane.
The primate-specific probe is added to the membrane and hybridization
of the probe to DNA on the membrane is detected by chemiluminescence.
In contrast to this labor-intensive method, new PCR-based quantification assays
provide automated detection and quantification of nucleic acid sequences. A human-specific
quantification assay is being developed based on real-time PCR amplification of
Alu sequences using TaqMan fluorescent chemistry (Holland et al. 1991;
Lyamichev et al. 1993). As part of this assay development, three existing human-specific,
real-time PCR-based quantification systems will also be evaluated (Applied Biosystems
2003; Nicklas and Buel 2003; Richard et al. 2003).
Integrated, Fully Automated System for Forensic Genotyping
A complete system for forensic nuclear DNA analysis could enhance
user convenience and relieve analysts from performing repetitive
tasks. The FBI is taking two approaches toward a totally automated
system. One is to implement robotic liquid handlers. The other is
to develop a miniaturized analysis system for forensic STR typing.
Robotic Liquid Handlers
Robotic liquid handlers will be programmed to integrate all of
the steps of forensic nuclear DNA analysis, from extraction of DNA
to profile analysis of the 13 core STR loci. Two robotic workstations
are planned for this task.
Figure 6. Robotic Liquid Handler for Performing the Manual
Steps of STR Typing
One of the workstations will perform DNA extractions, both differential
and nondifferential, and set up real-time quantitative PCR assays.
Using values from a real-time PCR system, this same workstation
will also prepare additional plates of DNA solutions at a common
concentration and set up PCR multiplex genotyping assays.
The second robotic workstation will prepare genotyping amplification
products for capillary electrophoresis analysis on a genetic analyzer.
In order to be compliant with quality assurance standards for forensic
DNA testing laboratories, the two robotic workstations will be
placed in separate rooms—a preamplification room and a postamplification
room, respectively (Federal Bureau of Investigation 2000A; Federal
Bureau of Investigation 2000B). Although this is not a fully automated
system, the robotic liquid handlers will dramatically reduce operator
involvement compared to the semiautomated steps described in the
Micro-Total Analysis System for STR Typing
A micro-total analysis system, also known as a lab-on-a-chip, is
a small (centimeter-sized) chip that contains micron-sized channels
for fluid transport and other design elements, such as pumps, valves,
and reactors that miniaturize, integrate, and automate complex,
multistep chemical and biological processes (Effenhauser and Mana
1994; Freemantle 1999; Lab-on-a-chip 2002; Manz et al. 1993; Meldrum
Compared to conventional laboratory instrumentation, micro-total
analysis systems are virtually hands-free, consume extremely small
volumes of samples and reagents, provide short sample-to-answer
times, and are inexpensive and small. The devices are constructed
from glass, quartz, silicon, or plastic using microfabrication technologies
from the semiconductor industry (photolithography, micropatterning,
microjet printing, light-directed chemical synthesis, laser stereochemical
etching, and microcontact printing), or casting, cutting, and stamping
The FBI has taken two approaches toward developing a micro-total
analysis system for forensic STR typing. The first combines all
of the functions required for forensic nuclear DNA analysis onto
one disposable chip. In this case, printed circuit boards will control
the on-chip heating and cooling required for thermal cycling, and
off-chip instrumentation will be used to achieve multicolor fluorescence
Figure 7. Prototype Micro-Total Analysis System for STR
The second approach for a STR-typing
micro-total analysis system is a modular system composed of three microchips—a
cell separation/DNA extraction chip, a multiplexed PCR chip, and a multichannel
electrophoresis chip. Microfluidic interconnects will transfer material from
one chip to the next, and off-chip systems will be used to actuate thermal
cycling and for multicolor fluorescence detection. In both micro-total analysis
system approaches, all required quality controls for STR typing will be accommodated
on the chip along with the samples.
Online Data Interpretation and Quality Assessment Tools
An enormous bottleneck in the process flow for forensic STR typing
is the interpretation of results and the associated data quality
review. Each forensic laboratory has established guidelines for
how these reviews are conducted. The guidelines include criteria
to determine whether the results are of sufficient intensity and
quality for interpretation, how to identify artifacts, and how to
interpret mixed DNA samples and partial profiles. Most laboratories
perform these functions manually, although software solutions are
emerging (Perlin 2000; Perlin et al. 2001).
The FBI is developing expert-system software for forensic STR applications
that will address single source and mixed samples. The software
will determine STR DNA fragment sizes relative to an internal size
standard, generate allelic designations, and translate the DNA signals
into useful information in accordance with a laboratory's interpretation
guidelines. The expert system will also assess the quality of the
data, note any problems, and put the data into a format compatible
for uploading into CODIS.
Figure 8. AmpFlSTR Profiler Plus STR Data Collected on
an ABI PRISM 310 Genetic Analyzer
Outsourced Research Projects
The FBI's research and development initiative to automate forensic
analysis of nuclear DNA is being executed primarily through external
contractors. Table 1 lists the projects that are part of this research
and development program.
Table 1: Research and Development Projects
|Source Attribution of Biological Evidence
|Microfluidic Differential Extraction Instrument
|Instrument and Automation Protocols for
Integrated DNA Extraction, Differential Extraction, Quantification,
Dilution, Amplification, and Set-up for Capillary Electrophoresis
Analysis of the STR Process
|Development of an Integrated STR-Analysis System
|Microfabricated Device for the Transfer
of Cells from a Swab to an Analytical Microchip
|Cell Separation and DNA Extraction on a Modular Microdevice
|DNA Quantification and PCR Amplification
on a Modular Microdevice
|Separation and Multicolor Detection of DNA on a Modular Microdevice
|Developing an Expert System to Interpret
Short Tandem Repeat DNA Results
Research and development for the automated nuclear DNA analysis initiative
began in July 2003, and deliverables from these projects are expected in 2005
or 2006. The first deliverables will increase automation of current STR-typing
procedures. The later deliverables will be fully automated lab-on-a-chip STR-analysis
Nineteen state and local forensic laboratories are participating
in this research and development effort through the FBI's Research
Partnership Program (Counterterrorism and Forensic Science Research
Unit 2004). Their involvement will include assisting in the evaluation
of prototype instruments, contributing data for training the expert-software
system, and conducting validation studies on the robotic liquid
handlers for STR typing, an automated sperm-searching protocol,
and the expert-software system.
For additional information contact
Kerri A. Dugan
Stephen T. Homeyer
Counterterrorism and Forensic Science Research Unit
Federal Bureau of Investigation
Applied Biosystems. Quantifiler Human DNA Quantification Kit
User's Manual. Applied Biosystems, Foster City, California,
Attorney General's Report on the DNA Evidence Backlog, April 2004.
Ballantyne, J. Serology: Overview. In: Encyclopedia of Forensic
Sciences. J. A. Siegel, P. J. Saukko, and G. C. Knupfer, eds.
Academic, London, 2000, pp. 1322-1331.
Caron, H., van Schaik, B., van der Mee, M., Baas, F., Riggins,
G., van Sluis, P., Hermus, M. C., van Asperen, R., Boon, K., Voute,
P. A., Heisterkamp, S., van Kampen, A., and Versteeg, R. The human
transcriptome map: Clustering of highly expressed genes in chromosomal
domains, Science (2001) 291:1289-1292.
Counterterrorism and Forensic Science Research Unit. FBI visiting
scientist program, Forensic Science Communications [Online].
(January 2004). Available: http://www.fbi.gov/hq/lab/fsc/backissu/jan2004/shortcomm/ 2004_01_short01.htm.
DNA Analysis Backlog Elimination Act of 2000, Pub. L.
No. 106-546, 114 Stat. 2726 (2000).
Effenhauser, C. S. and Mana, A. Miniaturizing a whole analytical laboratory
down to chip size, American Laboratory (1994) 26:15-18.
Federal Bureau of Investigation. Quality assurance standards for forensic DNA
testing laboratories, Forensic Science Communications [Online]. (July
2000A). Available : http://www.fbi.gov/hq/lab/fsc/backissu/july2000/codispre.htm.
Federal Bureau of Investigation. Quality assurance standards for
convicted offender DNA databasing laboratories, Forensic Science
Communications [Online]. (July 2000B). Available: http://www.fbi.gov/hq/lab/fsc/backissu/july2000/codispre.htm.
Freemantle, M. Downsizing chemistry: Chemical analysis and synthesis
on microchips promise a variety of potential benefits, Chemical
and Engineering News (1999) 77(8):27-36.
Gill, P., Jeffreys, A. J., and Werrett, D. J. Forensic application
of DNA fingerprints, Nature (1985) 318:577-579.
Holland, P. M., Abramson, R. D., Watson, R., and Gelfand, D. H.
Detection of specific polymerase chain reaction product by utilizing
the 5' to 3' exonuclease activity of Thermus aquaticus DNA polymerase,
Proceedings of the National Academy of Sciences of the USA
Juusola, J. and Ballantyne, J. Messenger RNA profiling: A prototype
method to supplant conventional methods for body fluid identification,
Forensic Science International (2003) 135:85-96.
Kellar, K. L. and Iannone, M. A. Multiplexed microsphere-based
flow cytometric assays, Experimental Hematology (2002)
Lab-On-A-Chip: The Revolution in Portable Instrumentation.
Technical Insights, Frost and Sullivan, San Antonio, Texas, 2002.
Lyamichev, V., Brow, M. A. D., and Dahlberg, J. E. Structure-specific endonucleo-lytic
cleavage of nucleic acids by eubacterial DNA polymerases, Science (1993)
Manz, A., Harrison, D. J., Verpoorte, E., and Widmer, H. M. Planar
chips technology for separation systems: A developing perspective
in chemical monitoring, Advances in Chromatography (1993)
Meldrum, K. Microfluidics-based products for nucleic acid analysis,
American Laboratory (1999) 31:20-22.
Nicklas, J. A. and Buel, E. Development of an Alu-based,
real-time PCR method for quantitation of human DNA in forensic samples,
Journal of Forensic Sciences (2003) 48:936-944.
Nolan, J. P. and Mandy, F. F. Suspension array technology: New
tools for gene and protein analysis, Cellular and Molecular
Biology (2001) 47(7):1241-1256.
Perlin, M. W. An expert system for scoring DNA database profiles. In: Proceedings
of the Eleventh International Symposium on Human Identification 2000. Promega,
Biloxi, Mississippi, October 2000. Available: http://www.promega.com/geneticidproc/ussymp11proc/content/perlin.pdf.
Perlin, M. W., Coffman, D., Crouse, C. A., Konotop, F., and Ban, J. D. Automated
STR Data Analysis: Validation Studies. In: Proceedings of the Twelfth International
Symposium on Human Identification 2001. Promega, Biloxi, Mississippi, October
2001. Available: http://www.promega.com/geneticidproc/ussymp12proc/contents/perlin.pdf.
Richard, M. L., Frappier, R. H., and Newsman, J. C. Developmental validation
of a real-time quantitative PCR assay for automated quantification of human DNA,
Journal of Forensic Sciences (2003) 48:1041-1046.
U.S. Department of Justice. The President's Initiative to Advance Justice
Through DNA Technology, Fact Sheet, March 11, 2003.
Walsh, P. S., Varlaro, J., and Reynolds, R. A rapid chemiluminescent
method for quantitation of human DNA, Nucleic Acids Research