2672 Maurice J. Aboud1,2 Marcus Gassmann1,2 Bruce R.

McCord1,2
1

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Research Article

Chemistry Department, Florida International University, Miami, FL, USA 2 R&D Assay Development, Agilent Technologies R&D Life Science Solutions Unit Liquid Phase Analysis Division, Waldbronn, Germany

The development of mini pentameric STR loci for rapid analysis of forensic DNA samples on a microuidic system
There is increasing interest in developing methods for portable DNA analysis in mass disasters and criminal identication. Currently most forensic STR DNA analysis is performed by CE; however, these instruments are not portable and require long sample run times. One potential solution is the development of microuidic systems for DNA typing. Unfortunately, fairly long (ca. 20 cm) separation channels are usually required for the proper resolution of multiplexed STR loci used in human identication. Commercially available systems like the Agilent 2100 Bioanalyzer have a small footprint and utilize chips with shorter channels and reduced resolution. Such portable systems might be valuable for evidence screening in remote locations. However, due to their lower resolution, most standard 4 base STR loci and their inherent 2 base variants will not resolve on such systems. In this paper, we discuss the development of reduced length pentameric (5 base) STR amplicons. Pentameric STRs have fewer variant alleles and are easier to separate due to the wider spacing between alleles. By incorporating novel denaturing sieving polymers in a short microuidic channel, we demonstrate efcient separations on these chips. Such an approach can serve as a useful tool for rapid microuidic DNA typing. Keywords: DNA / Forensics / Genotyping / Microuidics / Pentameric STR DOI 10.1002/elps.201000032

Received January 20, 2010 Revised April 11, 2010 Accepted April 14, 2010

1. Introduction
Although the use of traditional CE techniques has become standard for most forensic DNA analysis, there are some applications in which this larger instrument is not suitable due to cost, complexity, lack of portability and long sample run times (E30 min per sample). Mass disasters in remote locations have revealed a need for portable, user-friendly systems for screening DNA evidence (http://www.chemi. agilent.com/library/applications/5989-098SEN.pdf, accessed 06.02.10; Kupfer, D. M. et al., FAA Civil Aerospace Medical Institute 2006) In addition, many forensic laboratories are backlogged with DNA casework. Having a quick, reliable and accurate screening method would permit the analysis of mixed DNA evidence samples such as stains or allow investigators to piece together bone fragments and other similar forensic samples from a mass disaster. A related issue for such samples is the problem of sensitivity and recovery of degraded DNA. The recent development of

Correspondence: Dr. Bruce R. McCord, Chemistry Department, Florida International University CP 304, 11200 SW 8th St, Miami, FL 33199, USA E-mail: mccordb@u.edu Fax: 1305-348-3772

Abbreviation: HEC, hydroxyethyl cellulose

MiniSTR approaches has greatly assisted investigation of such samples [15]. As a result of the portability limitations of conventional CE, research and development into the application of forensic-based microchip electrophoresis has become widespread. These devices involve rapid separation of DNA in channels etched into glass chips followed by detection with laser-induced uorescence or amperometry. One of the rst applications of DNA separation on microuidic devices was demonstrated by Mathies and Woolley in 1995, where they were able to obtain single base pair resolution of DNA fragments between 150 and 200 bases in approximately 1015 min [610]. The decrease in separation time was due to the shorter length of the microchip and higher electric eld (http://www.chemi.agilent.com/library/applications/ 5989-098SEN.pdf, accessed 06.02.10). However, the DNA fragment sizes must be relatively small (100350 bp) in order to achieve high resolution. Microuidic devices can increase the sample throughput since multiple separation channels can be implemented on the same chip. Because they can be mass produced inexpensively from glass or plastic, these devices may be the key for quick and portable forensic applications [1114]. Maintenance and cross contamination problems are reduced due to the disposable separation platform. Microchip systems have been widely used in the analysis of a variety of different proteins and nucleic acids with good resolution and reduced sample run time. More specically, a
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number of investigators have examined microchip based electrophoretic systems for the separation of STRs and mitochondrial DNA [10, 1519]. While these devices are still at the prototype stage, the approach is feasible and ready for implementation. It is also clear that integrated approaches involving extraction, amplication and detection can be implemented on microuidic devices further reducing sample handling [2023]. One major issue with microuidic systems is developing devices with sufcient speed and resolution to compete with current CE technology. The separation mechanism used in microuidic devices (DNA sieving in entangled polymers) is the same as that required for standard capillary gel electrophoresis using 40-cm capillaries. While some gains in resolution may be obtained by microuidic systems due to their unique crossed T injections, there is still insufcient resolution in most systems to completely determine all 13 CODIS STRs and their 1- and 2-bp variant alleles. Instead, to properly resolve STR alleles, separation channels nearly 20 cm in length are required. Such long channels are awkward in chip-based devices, making them less portable and/or requiring serpentine geometries [6, 10, 22]. Truly portable devices should have much shorter channels to decrease their size and improve the analysis speed. Unfortunately, resolution, in turn, suffers. In previous research in the development of miniSTRs, we have noticed that shorter amplicons (60200 bp) require much less stringent conditions for their separation and are particularly valuable in the analysis of degraded and inhibited samples [3, 24]. Thus, these issues with short separation channels could be alleviated through the use of a smaller set of short amplicons that do not require such long separation channels. Such a sample set would be ideal for application with microuidic devices and would t the need for a rapid onsite screening tool [25]. This research has focused on the testing and adaptation of a commercially available microchip-based system for the analysis of miniSTRs based on a novel set of pentameric STRs. The use of pentameric nucleotide repeat units has been shown to reduce the amount of stutter in the amplied sample, a useful characteristic when dealing with mixtures. In addition, pentameric STRs are highly polymorphic and have relatively few microvariants, which make them ideal for forensic analysis. A multiplex consisting of three different pentameric STR loci was developed and tested using both CE and microuidic systems. The result was a robust and rapid system for screening and sorting forensic samples.

genome were obtained from the Promega Corporation while primers for the Penta D marker were taken from previously designed miniSTR loci [5].

2.2 Primer design PCR primers were designed for each marker using the web base Primer3Plus interface software [26]. Typically, the default primer parameters were used. The primers were designed as close to the target repeat unit to ensure that the smallest sized amplicons were used. An additional constraint on primer design was their annealing temperatures (Tm), which had to be compatible to permit multiplexed PCR reactions. The resulting primers and their sequences are listed in Table 1.

2.3 PCR primers and other reagents Fluorescently labeled (6-FAM, HEX and NED) primers, AmpliTaqTM Gold DNA polymerase and associated PCR buffers were obtained from Applied Biosystems (Foster City, CA, USA). The forward primers were labeled with 6FAM, HEX and NED dyes (Applied Biosystems), which permitted the use of compatible matrix standards with ABI Genetic Analyzers. Forward primers labeled with CY-5 were obtained from Integrated DNA Technologies (Coralville, IA, USA) and used for applications on the Agilent microchip system that has a red laser. All unlabeled primers were ordered from Integrated DNA Technologies. The design was intended to produce the smallest possible PCR amplicons for the three penta markers: Penta B, C and D. Smaller PCR products are less affected by degradation, amplify more efciently and can be better resolved. The three primer sets were prepared from the accession numbers such that the target region included the penta-nucleotide repeats and the primers were placed as close to the target region to minimize excess anking regions. A reduced size amplicon for the third marker, Penta D, had been previously developed and was not modied any further [5]. The fact that the three primer sets would be multiplexed was taken into consideration during the primer design phase of the research.

2.4 Polymer preparation The PCR amplied STR products were separated using several different denaturing buffer systems. Commercial POP-4 (Applied Biosystems), POP-6 sieving buffer (Applied Biosystems) and a custom denaturing polymer (PVP/ hydroxyethyl cellulose (HEC)) consisting of a 3.5% w/v mixture of polyvinyl pyrrolidone (Polysciences, Warrington, PA, USA) and HEC (Aldrich Chemical, Milwaukee, WI, USA) (20:80 ratio) were used for both the standard capillary system and the microchip assay [27]. A proprietary
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2. Materials and methods

2.1 Reference sequences and allele range information Reference sequences for the penta STR markers Penta B, Penta C and Penta D were obtained from GenBank. The location of the Penta B and Penta C markers within the
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Table 1. Penta markers and primer sequences Locus miniSTR primer sequence 50 to 30 Repeat motif AAAAG GTTTT AAAGA Fluorescent label (5) 6FAM (blue) Hex (green) Ned (yellow) Emission lmax (nm) 517 553 575 635 Excitation lmax (nm) 494 535 553 680 Tm (1C) 62 63 61 62 63 58 5 Tail added Distance from repeat (bp) 16 GTTTCTT 13 GTTTCTT 11 19

Penta B Penta C Penta D

F R F R F R

GAG GCA ACA GTG CGA GAC TTG AGC CTT GCA CTC CTA TT CAG GGA TAT GCA CTG GTA ATA GA CGC TTC TAG GGA CTT CTT CAG GAG CAA GAC ACC ATC TCA AGA A GAA ATT TTA CAT TTA TGT TTA TGA TTC TCT

denaturing polymer obtained from Agilent Technologies (Santa Clara, CA, USA) was also utilized for microchip separations.

2.5 DNA samples DNA standards from cell lines 9948 and K562 were purchased from Promega (Madison, WI, USA) and diluted to 0.11.0 ng/mL. These samples were used as the basis for the optimization of the STR multiplex. Anonymous buccal swab DNA samples were taken from a variety of subjects to provide a preliminary population study. These samples were approved for use through an institutional review board and were extracted using a standard phenol-chloroform/isoamyl extraction protocol.

2.6 Quantication of DNA samples All extracted samples were quantied using an Alu-based real-time PCR method with 0. 5 SBYRs-Green I dye (Molecular Probes, Eugene, OR, USA) [28]. Quantication was performed in reaction volumes of 20 mL using a Master Mix containing GeneAmps PCR Gold buffer (Applied Biosystems), 1.5 mmol/L MgCl2, 200 mmol/L deoxynucleotide triphosphates (Denville Scientic, dNTPs: dATP, dCTP, dGTP, dTTP), 1 mM BSA (Sigma-Aldrich, St. Louis, MO, USA), Triton X-100 (10% solution), Alu forward an reverse primers and two units of RampTaq hot start Taq polymerase (5 U/mL) (Denville Scientic, Metuchen, NJ, USA). A series of 9948 DNA standard solutions of known concentration were diluted ranging from 10 to 0.1 ng/mL and used to establish a standard curve. All samples were run on a Corbett Robotics Rotor Gene 6000 instrument (Qiagen Corbett Robotics, Valencia, CA, USA) and the software used to calculate the critical threshold values and calculated concentration of unknown samples [28].

200 mmol/L deoxynucleotide triphosphates (Denville Scientic, dNTPs: dATP, dCTP, dGTP, dTTp), 1 mM bovine serum albumin (BSA) (Sigma-Aldrich) and two units of AmpliTaq Golds DNA Polymerase (Applied Biosystems). Primer concentrations were adjusted from 0.5 to 1.5 mmol/L to optimize the amplication reaction and obtain balanced peak heights. A total of 0.5 ng of DNA template was used for all optimization experiments. Thermal cycling parameters, especially the annealing temperature, cycle number and extension times, were altered to establish the optimum amplication conditions using the GeneAmp 9700 thermocycler (Applied Biosystems), running samples on an ABI Prisms310 Genetic Analyzer (Applied Biosystems) and then compared for peak height and quality. The optimized thermal cycling conditions are shown below (denotes parameters that were varied during optimization, the cycle number (2834), annealing temperature (55601C) and extension soak time (1090 min). PCR cycling parameters are as follows: 951C for 10 min; 961C for 1 min; 32 cycles: 941C for 30 s,611C for 30 s, 701C for 45 s; 601C for 60 min; 41C for 10 min; 251C forever. The multiplexed amplication utilized 3 dye-labeled primers; 6FAM was used for Pena B, 6HEX for Penta C and NED for Penta D. A 50 -GTTTCTT- tail was added to the reverse primer of each locus to minimize possible problems with 7A non-template addition [5]. In addition, a nal soak at 601C for 60 min was added to the PCR cycle to minimize these effects.

2.8 Analysis on ABI 310 (single capillary) genetic analyzer The ABI Prisms310 Genetic Analyzer (Applied Biosystems) was used with lter set D to process the data from the four dyes 6FAM, 6HEX, NED and ROX after the matrix had been created using matrix standards from Applied Biosystems. Each sample was prepared by adding 1 mL of PCR product to 12 mL of Hi-DiTM formamide (Applied Biosystems) and 0.5 mL GS500 ROX size standard (Applied Biosystems). Samples were then placed immediately in the instrument for analysis. Samples were injected for 5 s at 15 000 V and separated at 15 000 V for 26 min with a run temperature of 601C. Standard electrophoretic conditions
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2.7 PCR amplication Amplication was performed in reaction volumes of 20 mL using a Master Mix containing 1 GeneAmps PCR Gold buffer (Applied Biosystems), 1.5 mmol/L MgCl2,
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were used including POPTM4 polymer, 1 Genetic Analyzer Buffer with EDTA (Applied Biosystems) and a 47-cm 50-mm capillary (Polymicro Technologies, Phoenix, AZ, USA). A small sample set was also analyzed using the PVP/HEC denaturing polymer buffer. Peak sizing was performed using the Global Southern algorithm.

2.9 Analysis with microuidic electrophoresis Two different systems were used for the microuidic analysis of the mini penta STRs, a standard Agilent 2100 Bioanalyzer (Agilent Technologies) for use with native DNA and a beta version of the same system with an adjustable heat plate capable of performing denaturing analysis. Native DNA separations were performed using the standard 2100 system with the DNA 1000 Lab-on-Chips Assay kit (Agilent Technologies) following the standard protocol provided. The chip was rst primed with the gel dye mixture and each sample for analysis was prepared by adding 1 mL of CY-5 uorescently labeled PCR product and 5 mL Agilent Marker (Agilent Technologies) to each of the 12 sample wells. The Agilent DNA 1000 ladder (Agilent Technologies) was used as a sizing standard. The results were processed to determine fragment size based on the sizing ladder and internal standards, using the manufacturers software provided with the system. Samples run using denaturing conditions were analyzed in a similar fashion except that a heated electrophoretic platform at 601C was used and the chips were modied with wider central channels to reduce problems with the increased viscosity of the buffer. When prepared for analysis using the denaturing chip, the sample is diluted in formamide prior to analysis. The PVP/ HEC polymer described above and a denaturing polymer supplied by Agilent Technologies was used for testing of the system and separation of alleles. The denaturing polymers were much more viscous than that of the existing DNA 1000 assay polymer and required careful pipetting and priming of the chip to ensure that the polymer was distributed throughout all the channels. Sizing was performed using two standards, an 8-bp lower marker and a 600-bp upper marker. In addition, a specic sizing ladder provided by Agilent Technologies was used to calibrate the separation system.

[29, 30]. These combined factors should make a pentameric multiplex ideal for rapid separations using short channel microuidic chips. The separations performed in this project took place on standard ABI 310 Genetic Analyzer with a 40-cm capillary and on the Agilent 2100, a commercially available DNA microchip system with a 3-cm separation channel. In its standard native DNA separation mode, the Agilent system provides resolution in the range of 612 bp depending on the DNA fragment size (http://www.chemi.agilent.com/ library/applications/5989-098SEN.pdf, accessed 06.02.10) [31]. While there have been a number of reports of the application of the system with standard forensic STR systems, this level of resolution is insufcient for regular application to 4-bp STR repeats, especially given the fact than many CODIS loci have common 2-bp variants [32, 33]. For this reason, we became interested in the pentameric STRs. To achieve maximal resolution of these loci, we also tested a new beta version of the Agilent 2100 chip system with an on-board chip heater that permits the use of denaturing buffers for enhanced resolution. It also should be noted that the detection system for this instrument is presently single channel, so multiplex detection based on different uorescent dye labels is not possible. However, the chip system does provide a reliable platform to test assumptions about effective resolution and throughput of short channel microuidic chips. The ABI 310 CE system was then used to demonstrate the ability to amplify the three pentameric markers simultaneously in a multiplex. The pentameric multiplex was developed using STR locations provided by Promega Corporation. The design produced the smallest possible PCR amplicons for three different pentameric markers. The primer sequences were selected using Primer 3 software and the PCR reaction conditions were then optimized for each individual locus using the ABI 310 CE system [26, 33].
9000 8000 7000 6000 5000 4000

3. Results and discussion

The goal of this project was to develop a set of reduced size pentameric STR markers for use in microuidic systems. The hypothesis was that mini STRS should provide enhanced sensitivity with these systems, permitting the rapid sorting and identication of forensic specimens in the eld. In addition, since pentameric alleles are farther apart than the standard four base repeats used for the 13 CODIS markers, resolution of mixtures and heterozygous alleles should be less of a problem. Pentameric repeats also have fewer two base variants than four base STRs and less stutter
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3000 2000 1000 0

0.5
Bm Allele 1

0.75
Bm Allele 2

1
Cm Allele 1

1.25
Dm Allele 1

1.5
Dm Allele 2

Figure 1. Graph of primer optimization showing the allelic height (RFU) versus the nal primer concentration (mM). The primer concentration with the highest peak heights and most balanced alleles between all three loci occurs at 1.5 mM. Bm, Cm and Dm correspond to the respective mini-penta Loci. All experiments were performed using 0.5 ng of template and 32 cycles of amplication. Other conditions are as listed in the Section 2.

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Figure 2. Electropherogram from ABI 310 Genetic Analyzer of 0.5 ng template K562 DNA Standard, lter set D, injection 5 s at 15 kV and run temperature of 611C. Lane (A) Penta Bm Marker Alleles at 124.06 and 138.86 bp. Lane (B) Penta Cm marker alleles at 107.55 and 118.35 bp. Lane C Penta Dm marker alleles at 118.35 and 138.79 bp.

To ensure selectivity, and allele balance of the PCR reactions, a number of parameters were optimized, including annealing temperature (55651C), primer concentration (0.51.5 mmol/L) and extension times (090 min). Based upon the theoretical primer melting temperature, it was determined that the best annealing temperature for the multiplex system was between 60 and 621C. These conditions minimized non-specic amplication and A peaks. For the primer concentrations, the goal was to optimize sensitivity and peak balance (Fig. 1). Experiments were performed examining the overall primer concentrations as well as individual concentrations within the different loci (all other conditions were as stated in the Section 2). The optimum multiplex conditions were observed at an annealing temperature of 611C and nal primer concentration of 1.5 mM using 0.5 ng of template and 32 cycles of amplication. A measurement of average allele size for Penta B produced a standard deviation of 70.08 bp while Penta C and Penta D had slightly higher standard deviation values of 70.15 bp and 70.13 bp, respectively. These deviations are most probably due to small temperature uctuations and slight mobility shifts during each run on the ABI Prisms310 Genetic Analyzer. Figure 2 is an example of the multiplexed amplication using the K562 DNA standard run under these conditions. To test the validity of these conditions, a small set of 20 individuals were analyzed using these conditions. The results showed consistent and reproducible amplication with the average stutter values of less than 6% for Penta B and under 1% for Penta C and Penta D.

3.1 Analysis by microuidic chip The currently available conguration of the Agilent 2100 Bioanalyzer involves uorescent detection of dsDNA using an intercalating dye for uorescence detection. Native, dsDNA is inherently more difcult to separate due to its increased rigidity (45 nm persistence length) when compared to single stranded DNA (4 nm persistence length). This difference in exibility affects the mobility of the DNA through the sieving polymer matrix and hence its separation [21]. Thus, the resolution of this dsDNA chip (68 bp) is limited mainly to applications in which agarose gels are commonly used. Given this fact, it is very difcult to separate 4-bp STRs using these devices. However, separation of pentameric repeats was thought to be possible. Figure 3 illustrates the separation of the Penta D and the mini- Penta D marker under these conditions. The results demonstrate a resolution of 12 bp for the large Penta D marker while the mini-penta D marker had a 9-bp resolution [34]. When the larger Penta D marker and the mini-Penta D marker were amplied and run on the Agilent Bioanalyzer chip, there was a clear improvement seen in the resolution of the two peaks with the mini primer (Fig. 3). This system used a nondenaturing polymer allowing for dsDNA separation with an intercalating dye. The enhanced resolution of the smaller allele set is attributed the fact that smaller DNA is less affected by orientation in the electric eld and polymer dynamics [35].

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Figure 3. Result from Agilent Bioanalyzer 2100 electropherogram of a K562 DNA standard showing the penta D alleles 9 and 13 on 1.3-cm separation channel length run at 350 V/cm. Top panel large Penta D marker with K562 standard alleles 9 and 13 (20 bp apart). Bottom panel the mini-Penta D marker (Dm) with reduced amplicon sizes and improved resolution.

490,500bp

490,500bp

490,500bp

139, 150,160bp 139, 150,160bp 139, 150,160bp

Figure 4. Results from Agilent Bioanalyzer 2100 comparing, ABI POP-6, 3.5% PVP/HEC custom denaturing polymer and Agilent denaturing polymer on a 1.3 cm separation channel length run at 350 V/cm. (Note that time scales (s) vary due to differences in polymer separation time)

Although there was a signicant improvement by using smaller sized PCR products in the native polymer, the resolution still was not sufcient to fully resolve peaks differing by 5 bp. To provide resolution enhancement, the development of a denaturing DNA separation was undertaken using a mixed PCP/HEC polymer with 8 M urea [27]. We also began working with a modied Agilent Bioanalyzer 2100 that contained an improved chip design and a heat plate. The added heat plate helps maintain the DNA sample in a denatured state. We then examined the system resolution under these new conditions using both the newly developed polymer and a standard POP-6 polymer
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obtained from Applied Biosystems. As can be seen in Fig. 4, the PVP/HEC polymer provides improved resolution for smaller sized DNA fragments when compared to a commercial ABI POP-6 polymer. This is seen by a clear baseline resolution of the 139, 150 and 160 bp size fragments of a LIZ 500 DNA sizing standard (Applied Biosystems). A commercial polymer system obtained from Agilent technologies was also evaluated. It provided further resolution enhancement. The combined effects of the smaller amplicon size, denaturing polymer and pentameric STR markers allowed for a system that could clearly distinguish adjacent
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Figure 5. ssDNA assay showing Penta B, C and D Markers of an extracted DNA sample 29. Top Penta Bm showings alleles at 118 and 128 bp with a resolution of 2.2 bp. Middle Penta Cm showing alleles at 111 and 121 bp with a resolution of 2.2 bp. Bottom Penta Dm showing alleles at 124 and 144 bp with a resolution of 2.2 bp. Data are reported in terms of size (base pairs); total run time is under 2 min.

alleles (5 bp) at baseline resolution (Fig. 5). The average resolution for each loci of the dsDNA assay for Penta Bm, Cm and Dm were 7.5, 8.3 and 8.5 bp (data not shown), respectively, while the resolution increased to 2.2 bp for all three of the ssDNA assay loci. The resolution increased on average about 3.7 times greater for the ssDNA assay. The shorter micro-channel also provided much faster separations with a sample separation time of less than 2 min.

an average resolution increase of about 3.7 times between loci using the faster, shorter micro-channel system. The mobility of ssDNA and the larger sized penta repeats allows for the required resolution and added advantages of less stutter and fewer variants especially when dealing with mixtures and degraded forensic samples. This allows for a fast DNA separation on a microchip with high resolution of the larger penta STRs and aid as a viable screening tool for forensic DNA analysis. The authors thank Agilent Technologies for their major support and supplying a Bioanalyzer prototype system. They also thank Dr. Len Goren and the Promega Corporation for their continued support and aid in the design of the pentameric STRs and Dr. George Duncan for his careful review of this work and helpful advice. The authors have declared no conict of interest.
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4 Concluding remarks
It has been shown that the use of pentameric STR markers with smaller amplicon sizes and a denaturing polymer for ssDNA separation on a microchip system is capable of resolving between alleles and can act as a quick onsite screening tool for forensic DNA analysis. This is shown by
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