Portable Devices For Possible Crime Scene Investigations

Microfabrication techniques revolutionized the integrated circuit industry 20 years ago and have brought the world ever faster and more powerful computers. These same microfabrication methods are now being applied to develop miniature, microchip-based laboratories, or so-called 'labs-on-a-chip' (Paegel et al. 2003). Miniaturizing the sample preparation and analysis steps in forensic DNA typing could lead to devices that permit investigation of biological evidence at a crime scene or more rapid and less expensive DNA analysis in a more conventional laboratory setting. We will focus here on three areas of on-going research for miniature DNA analysis instruments: microchip capillary elec-trophoresis (CE) devices, miniature thermal cyclers, and hybridization arrays.

Microchip CE Devices

The primary advantage of analyzing DNA in a miniature CE device is that shorter channels, or capillaries, lead to faster DNA separations. Instead of using a 30 cm long glass capillary tube to perform the DNA separation, microchip CE devices are typically glass microscope slides with narrow channels etched into them that are 10-50 |lm deep by 50 |lm wide and several centimeters long. A glass coverplate is bonded on top of the etched channels in order to create a sealed separation channel (Woolley and Mathies 1994). Alternatively, injection molded plastic may be used (McCormick et al. 1997).

Separation speeds that are 10-100 times faster than conventional elec-trophoresis may be obtained with this approach. Using a 2 cm separation distance (compared to 36cm for an ABI 310 capillary), tetranucleotide short tandem repeat (STR) alleles were separated in as little as 30 seconds (Schmalzing et al. 1997). Microchip CE systems are being developed with multi-color detection formats. These systems should therefore be compatible with commercially available STR kits. Figure 17.1 shows a simultaneous two-color analysis of polymerase chain reaction (PCR) products from the eight loci in the PowerPlex™ 1.1 STR kit (Schmalzing et al. 1999). This separation was performed in less than 21/2 minutes.

In order to obtain ultrafast DNA separations, the injection plug must be narrow and short compared to the separation length. DNA separations in less than 30 seconds have also been demonstrated with short capillaries using a fast ramp power supply for rapid injections (Muller et al. 1998). Research is ongoing to improve separation speeds and ease of use with the hope that in the near future microchip CE devices will be used routinely for rapid DNA analyses (see Mitnik et al. 2002, Goedecke et al. 2004).

A capillary array microplate device has been constructed with 96 separation channels in order to scale up the number of samples processed at a single time. These devices are capable of separating 96 different samples in less than two minutes (Shi et al. 1999). Although the DNA separation parameters need

Separation Time (minutes)

Figure 17.1 Rapid microchip CE separation of the eight STR loci from PowerPlex 1.1 (Schmalzing 1999). The electropherograms from scanning each color of a simultaneous two-color analysis are divided. The PCR-amplified sample is mixed with the allelic ladders prior to injection to provide a frame of reference for genotyping the sample. The allele calls for each locus are listed next to the corresponding peak. Figure courtesy of Dr. Daniel Ehrlich, Whitehead Institute.

improvement before this particular 96 channel microplate can resolve closely spaced STR alleles, the device demonstrates that rapid DNA separations are feasible in a highly parallelized format. Separations of STR alleles that have been demonstrated to date on multi-channel microchip devices (see Medintz et al. 2001, Mitnik et al. 2002, Goedecke et al. 2004) are performed at speeds that are really no different than what can already be accomplished with the conventional capillary array electrophoresis instruments described in Chapter 14.

Miniature Thermal Cyclers

Sample preparation devices are also shrinking in size (see Paegel et al. 2003). In particular, miniature thermal cyclers are being developed for performing PCR. These devices are being microfabricated with silicon reaction chambers (Northrup et al. 1998). A major advantage of miniaturizing the PCR thermal cycling process is the potential for lower reagent consumption and thus reduction in the cost of an analysis. In addition, more rapid thermal cycling times are possible because the PCR reaction mixture can be heated and cooled quickly. Since the reaction volume is smaller, it takes less time to thermally equilibrate the PCR reaction.

The miniature analytical thermal cycler instrument (MATCI) developed at Lawrence Livermore National Laboratory weighs only 35 pounds, fits in a medium-sized briefcase, and is powered by 13 rechargeable batteries. The MATCI system has been used to successfully amplify DQA1 alleles and a STR triplex consisting of the D3S1358, VWA, and FGA loci (Belgrader et al. 1998). A 25 ||L PCR reaction was performed with 30 cycles in about 42 minutes using MATCI compared to 2.5 hours on a PE9600 thermal cycler. The MATCI has also been used in conjunction with time-of-flight mass spectrometry to complete a STR genotyping assay in less than 50 minutes (Ross et al. 1998).

Ultimately, the combination of sample preparation in a miniature thermal cycling device coupled to rapid DNA analysis on a microchip CE device may be the future (Lagally et al. 2001). There are certainly a number of applications for which developed capabilities of highly rapid human identification would be of value, such as biometrics (D.N.A. Box 17.1). In the first demonstration of coupling a microfabricated PCR reactor with a micro-capillary electrophoresis chip, a rapid PCR-CE analysis was performed in less than 20 minutes (Woolley et al. 1996). In this particular case, a PCR amplification of a single amplicon involving 30 cycles was performed in 15 minutes and was immediately followed by a high-speed CE chip separation in 83 seconds.

A similar type of online, automated DNA amplification and separation has also been demonstrated with a larger scale CE system (Swerdlow et al. 1997). In this case, the total time from extracted DNA to result was 20 minutes - eight minutes for thermal cycling, four minutes for purification, and eight minutes for electrophoresis. It may well be that in the not too distant future DNA results, from biological sample to STR profile, may be routinely obtained in under an hour. This type of rapid analysis would then enable DNA testing at or near crime scenes in mobile laboratories.

One major obstacle to seeing this type of rapid analysis achieved is that current STR multiplexes are not compatible with rapid thermal cycling. Rather, well-balanced amplification yields with the primer concentrations present in commercial STR kits are only achieved when ramp rates of 1°C per second are used rather than 5-10°C per second involved in rapid cycling.

STR Determination by Hybridization Arrays

Nanogen Inc. (San Diego, CA) has developed another microchip-based assay that initially appeared promising for rapid STR allele determination. However, this approach is no longer being actively pursued by forensic DNA laboratories for STR typing.

The field of biometrics involves developing automated methods of identifying a person or verifying the identity of a person based on a physiological or behavioral characteristic. Current modalities of biometrics include fingerprints, iris scans, hand and finger geometry, face recognition, voice recognition, and signature verification. So called 'smart cards' are being developed to provide increased security for a variety of applications. Biometric information is also being included on passports to help prevent falsification of passport identity.

Biometric authentication requires comparing a reference sample that is collected during the 'enrollment' phase against a newly captured biometric sample collected as part of identification. Access to a particular location or computer file is based on whether a match can be verified between the identification biometric signal and anyone of the authorized signatures (e.g., fingerprints, voice pattern, etc.) collected during enrollment.

The 1997 movie GATTACA is a futuristic vision of a world where rapid genetic testing is used to prevent access to secure locations by genetically 'imperfect' individuals. Currently this concept of using DNA for a biometric is still futuristic, primarily because of the cost and length of time required to perform DNA testing. However, as DNA analysis because less expensive and more rapid, it may become a valuable biometric to be used beyond the national DNA databases discussed in Chapter 18.




D.N.A. Box 17.1 DNA and biometrics

The Nanogen assay involves the use of a silicon microchip composed of an arrayed set of electrodes that act as independent test sites (Figure 17.2). Electric potentials can be directed to each test site, which contains a unique DNA probe for hybridization (Sosnowski et al. 1997a).

A DNA hybridization assay is conducted by washing a DNA sample over the chip and seeing where it binds on the array. PCR-amplified samples will bind or hybridize to their complementary probe sequence. An 'electronic stringency' can then be applied to each probe site by simply adjusting the electric field strength. Samples that are not a perfect match for the probe will be denatured and driven away from the probe.

Since each test site is separate from the others on the spatial array of probes, a signal obtained from a particular position on the probe array indicates which sequence has bound to the probe. Fluorescent probes may be added to the DNA molecules for detection purposes. Software then reads the array position versus fluorescent signal and interprets the data to determine the sequence present in the DNA samples being measured.

In order to measure which STR alleles are present at a particular locus, a chip is prepared that contains known alleles for that locus. Each probe position on

Figure 17.2

Schematic of nanogen STR hybridization chip assay. The assay illustrated in (a) involves a capture probe oligonucleotide that is attached at a unique location on the chip (b). The capture probe hybridizes to the appropriate STR allele by binding to the repeat region and 30-40 bases of the flanking region. A reporter probe containing 1-3 repeat units, some flanking sequence and a fluorescent dye hybridizes to the STR allele and generates a fluorescent signal at the probe site that can be interpreted to yield the sample's genotype (c). Multiple STR loci may be probed on the same chip with one probe site existing for each allele.

STR allele containing 6 repeat units

Hybridization between probes and STR file

STR allele containing 6 repeat units

Capture Probe (attached to chip)

Reporter Probe (fluorescent dye attached)

Capture Probe (attached to chip)

Reporter Probe (fluorescent dye attached)

Alleles (Repeat #)

Locus A





Locus B





Locus C





Locus D




Locus A = 6,8 Locus B = 3,4 Locus C = 6,8 Locus D = 6,6

the chip has a different allele attached (Figure 17.2b). Thus, in order to have the capability of measuring eight alleles at the TPOX locus (e.g., 6-13 repeats), eight different probe sites are required.

The STR hybridization assay involves two probes that bind to the STR repeat and flanking regions. The 'capture probe' is attached to the chip at the test site and captures the PCR-amplified STR allele when the sample is added to the chip. The 'reporter probe' contains the fluorescent dye and thus enables the detection of the STR allele bound to the particular site defined by the capture probe (Figure 17.2a). The unique sequences on either side of the repeat make it possible to have discrimination between alleles of different STR loci with the same repeat sequence.

A sample's genotype is assessed by observing the positions that give a fluorescent signal once the PCR product has hybridized to its corresponding capture probe(s). The read-out provides a genotype that corresponds to the number of repeats present in the sample even though no size-based separation has been performed (Figure 17.2c). Thus, samples measured with this hybridization assay can be compared to results obtained from a conventional DNA separation of the STR alleles. Nanogen has conducted several successful sample correlation tests with the Bode Technology Group, a private forensic laboratory located in Springfield, Virginia (Sosnowski et al. 1997b).

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