Detectives investigating crime scenes often find biological evidence such as blood or hair that can be used to prove the guilt or innocence of suspects. Examining this evidence using DNA analysis, criminologists can determine whether a suspect was present at the crime scene. In the biology/serology laboratory of the Metro-Dade Police Department (Dade County, FL), investigators are using vision-based DNA fragment analysis and comparison systems to perform this examination.

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By John Haystead, Contributing Editor

Detectives investigating crime scenes often find biological evidence such as blood or hair that can be used to prove the guilt or innocence of suspects. Examining this evidence using DNA analysis, criminologists can determine whether a suspect was present at the crime scene. In the biology/serology laboratory of the Metro-Dade Police Department (Dade County, FL), investigators are using vision-based DNA fragment analysis and comparison systems to perform this examination.

In the laboratory, both Restriction Fragment Length Polymorphism (RFLP) and Polymerase Chain Reaction (PCR) techniques are used to analyze DNA molecules. While RFLP is used to identify gene sequences by measuring the relative size and locations of individual DNA fragments, PCR is used where only small amounts of biological evidence exists.

Parallel strands

DNA molecules are composed of two parallel strands of four bases, adenine (A), guanine (G), cytosine (C), and thiamine (T), configured in a double helix. Each base is paired to its counterpart on the corresponding string according to a specific pattern, A with T, C with G.

Once extracted from its source, DNA strands are chemically cut into small pieces by an enzyme that recognizes specific sequences of base pairs in the DNA molecule. This restriction digestion process results in a large set of DNA fragments of varying lengths. These fragments are then placed at one end of an 8-cm block of semiporous gel for electrophoresis.

Because DNA fragments have a negative charge, they are pulled through the gel as current is applied. Because smaller fragments travel faster than larger ones, the result is a spectrum of DNA fragments distributed across the gel. Because the gel is fragile, the DNA is then transferred onto a tough nylon membrane using a blotting process. At this point, the two parallel strands of DNA in each fragment are also separated or "denatured" to break the individual base pairs.

After the denaturing process, the membrane is dipped into a solution containing pieces of synthetic denatured DNA or "probes" that combine with the DNA being tested. The probes are composed of specific sequences of bases designed to pair with specific parts of human DNA known to exhibit a high degree of polymorphism, or size variation, across individuals. The probe DNA also incorporates a radioactive label or marker that can be read on an x-ray film or "autorad." Multiple probes are used to mark the DNA, generating a complete DNA profile of individuals. Radioactive markers on the DNA appear as dark images, producing either one large or two distinct spots for each marker.

Digitizing film

The autorads are placed on a light box with a CCD television camera overhead. Images are digitized using a PC-based frame grabber from Data Translation (Marlboro, MA) that is housed in a Pentium PC. Here, custom windows-based software developed by the FBI (Washington, DC) extrapolates the size of the DNA fragments by comparing them against standard reference markers interspersed among the DNA samples.

Written in Visual BASIC, the Combined DNA Index System (CODIS) software creates a densitometric scan of the image and marks the peaks at each band from which it calculates the molecular weight of each fragment. Metro-Dade criminologist Monroe Chin-see has written additional software functions for CODIS that the FBI has incorporated into its DNA analysis system. DNA databases of known felons are maintained at state and federal laboratories and can be searched using dial-up connections over encrypted telephone lines.

According to Chin-see, the FBI set out to make the CODIS system as affordable as possible using standard off-the-shelf equipment wherever possible. Now, Chin-see is evaluating more sophisticated systems and software to interface with the program. One such system is the Bio Image (Ann Arbor, MI) DNA sequence film reader and sequence assembly manager.

The Bio Image system replaces the CCD camera with a 12 ¥ 17-in. Mirage color scanner from Umax (Fremont, CA). The scanner features a 400 ¥ 800-dpi resolution and 10-bit A/D output, and 3.2 dynamic range is hosted by a SUN Sparc 4 workstation. With an attached 2.6-Gbyte optical drive, the system can handle up to 1000 individual sequences automatically, accounting for band thickness, image intensity, and lane-to-lane loading variations. Operators also can select color-annotation schemes from a 36-color palette and generate 3-D plots and image histograms. "Compared to the 512 ¥ 512-pixel resolution of the CCD system, the higher resolution provided by the digital scanner is very beneficial in assigning size values to the autorads," says Chin-See.

Tiny samples

At some crime scenes, the limited amount of biological evidence means that RFLP techniques cannot be used. Here, PCR is used to target individual areas on the genome and amplify them by synthesizing (or cloning) that portion of the DNA. Target sites are much smaller than those measured by RFLP and have a high degree of polymorphism or variance between individuals.

The PCR technique starts with a PCR "cocktail" comprised of denatured source or "template" DNA, polymerase enzyme, and a mixture of the four DNA bases that have fluorescent die molecules attached to them. The cocktail is placed in a temperature-programmable thermal cycler where the polymerase enzyme synthesizes a replica of the template DNA incorporating the die-marked base molecules. The process is repeated many times until an adequate amount of synthetic DNA is produced.

Amplified DNA is then placed on a gel and placed into an ABI PRISM 377 DNA sequencer from Perkin-Elmer (Norwalk, CT) for electrophoresis and analysis. As the DNA fragments pass through the instrument, an argon-ion laser excites the dyes at 488 and 514 nm. The emitted light then passes through four color filters and onto a detector that outputs an image similar in appearance to an x-ray film autorad but with greater detail and enhanced with computer-simulated color. Electrophoresis separation can be viewed on-screen in real time with final data printed in a variety of formats. The ABI-377 is controlled by an Apple Power Macintosh 7100. "In addition to high throughput," says Chin-see, "an internal size marker within each DNA electrophoresis lane eliminates the need to extrapolate from reference markers."

Other tools needed

In addition to DNA analysis, the Metro-Dade laboratory uses a number of other image-processing tools and specialized analysis software for a range of trace-evidence analysis (see " Refractive index measurement identifies glass fast," p. 24, and "Forensic scientists access microscopes remotely," p. 25). One area where there is a continuing need for new image-based technology, however, is in the drug-analysis unit.

Although, the Metro-Dade analysis unit handles more than 12,000 cases a year involving an extensive array of controlled substances, analysts must visually identify each drug compound by examining its crystalline structure and chemical reactions under a microscope. Jose R. Almirall, senior criminalist in the Metro-Dade Police Department crime laboratory, says, "I don`t know of any instrument out there yet that can automatically recognize the shapes of these crystals and identify their type based on the chemical reactions. Such a system could also store images for court presentations. Now we photograph the slides and take Polaroid snapshots to court."

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In the crime laboratory of Florida`s Metro-Dade Police Department, forensic scientists are using DNA analysis to match criminal evidence with DNA profiles of felons.

Refractive-index measurement identifies glass fast

Different glasses exhibit a range of refractive indexes (RIs) based on the raw materials and manufacturing processes used to create them. For example, automobile-headlight glass (borosilicate glass) has a low RI compared to leaded ornamental glass. Analysts at the Metro-Dade Police Department (Dade County, FL) are using the RI of glass to identifies sources of glass fragments with a GRIM2 glass refractive-index measurement system from Foster & Freeman (Esham, Worcestershire, England).

With the GRIM2, glass fragments are immersed in a silicon oil bath built by Mettler-Toledo Process Analytical (Wilmington, MA). Because the RI of the oil changes with temperature, the hot stage heats the oil bath from room temperature to 110C, until the RI of the oil matches that of the glass fragment and the glass is no longer visible.

To observe the process, the system is equipped with a 10X phase-contrast microscope integrated with a CCD video camera from Sony Electronics (Park Ridge, NJ). Images are then digitized into a 486AT PC using a PC-based frame grabber.

In operation, the system continuously monitors the hot stage and automatically detects and records the point of minimum contrast between the glass and oil. When this occurs, the temperature is recorded and translated into a refractive-index value for the particular glass being analyzed. Because the system automatically determines the RI match point, it eliminates the need for visual assessment and reduces the possibility of human error. Repeat measurements on the same glass fragment have a standard deviation of 0.00002RI over a five-hour period and 0.00003RI over a five-day period.

The GRIM2`s Glass for Windows (GFW) software controls the hot stage and prompts the operator to collect, process, and present data. A graphics-based software program called the Fragment Data System developed by the Forensic Science Service (Birmingham, England) can be interfaced to the GFW software to organize large quantities of refractive-index data into specific, predetermined formats, or populations for follow-on statistical analysis.

Because of the number of variables involved, Jose R. Almirall, senior criminalist in the crime laboratory of the Metro-Dade Police Department, doesn`t believe RI information alone will ever specifically identify individual glass manufacturers. However, "the tool is extremely effective at excluding many sources and can significantly narrow down the field of possibilities," he says. Follow-on chemical analysis using an inductively coupled plasma instrument determines the specific trace elements in glass fragments.

J. H.

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To measure the refractive index of glass, samples are heated in an oil bath (above) until the refractive index of the oil matches that of the glass fragment and the glass is no longer visible. Images of glass fragments under test (inset) are stored for later analysis or database retrieval.

Forensic scientists access microscopes remotely

Without leaving their desks, forensic analysts can now remotely access and control transmission and scanning electron microscopes (TEM/SEMs) at Oak Ridge National Laboratory (Oak Ridge, TN). The laboratory is already providing remote access to both its Hitachi HF-2000 field emission TEM and S-4500 field emission SEM. Each microscope is controlled by two PowerMAC 9500 computers running DigitalMicrograph microscope-control software from Gatan (Pleasanton, CA).

Remote users can take control of DigitalMicrograph from their own computers using Timbuktu Pro software from Farallon (Alameda, CA). Analysts send their samples to the laboratory in advance and schedule a time for their remote session. They then dial up the TCP/IP protocol address of the control computer and receive an image of the Macintosh desktop at their PC.

According to senior research staff member Larry Allard, the biggest challenge to widespread use of the capability remains bandwidth. To overcome this, the laboratory has multiplexed four ISDN lines to provide direct point-to-point access at 512-kbit/s rates. With an additional ISDN line, remote users can also conduct video teleconferencing or "telepresence microscopy" using inexpensive cameras and See You See Me software from Cornell University (Ithaca, NY).

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Senior research staff member Larry Allard (seated) appears to be demonstrating the capabilities of the Hitachi HF-2000 electron microscope at Oak Ridge National Laboratory. But, the $1.6 million field-emission gun transmission electron microscope is being operated by a remote user.

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