MICROFLUIDIC VOLUME REDUCTION SOLID PHASE EXTRACITON OF
COMPROMISED AND LOW DNA TEMPALTE FORENSIC SAMPLES
C.R. Reedy1, J.J. Higginson1, and J.P. Landers1, 2
1
University of Virginia, USA
University of Virginia Health Sciences Center, USA
2
ABSTRACT
Microfluidic devices are ideal for accommodating low volume samples for extraction of DNA, but large volume samples (0.5 – 3 mL) challenge microfluidic devices that typically handle microliter volumes. Large volume samples are
encountered in forensic casework samples collected from surfaces, bone and teeth which often require milliliters for adequate demineralization, and diluted samples (to minimize impact of contaminants) which also dilutes the DNA template
in the sample. This work describes a volume reduction solid phase extraction (vrSPE) microdevice capable of handling
large volume samples that contain dilute concentrations of DNA, and its application to degraded and low template samples.
KEYWORDS: Solid phase extraction, DNA, degraded forensic samples
INTRODUCTION
Microfluidic solid phase extractions (µSPE) have proven to be reproducible and efficient for DNA purification using
silica-based solid phases [1]. However, the methods developed thus far are ideal for processing low volume samples as
opposed to large volume samples (0.5 – 3 mL) which are often encountered in forensic casework. These sample volumes are seen when collection from a surface is required and when the sample must be diluted to minimize the impact
of contaminants that inhibit polymerase chain reaction (PCR) [2]. This dilution also results in ‘low template’ samples
because as contaminants are diluted, so is DNA. Low template samples can also be encountered when environmentallydegraded evidence is collected, as the sample may still require solubilization from a surface, and from samples such as
bone or teeth samples that require milliliters for adequate solubilization [3]. Thus, a microdevice capable of volume reduction solid phase extraction is necessary when these samples are encountered.
THEORY
Although µSPE has been demonstrated successful for the purification of DNA from biological samples, little has
been shown for the application to compromised or degraded samples, which accounts for the majority of samples analyzed by the forensic community. Additionally, use of standard µSPE chip designs for large volume sample processing
would require loading times in excess of 2 hours, in addition to the subsequent extraction steps (washing to remove cellular and extracellular material and elution of the DNA), thus, negating one of the advantages of microfluidics, decreased
analysis time. To solve these issues, a volume reduction solid phase extraction (vrSPE) method is necessary.
EXPERIMENTAL
A vrSPE microdevice (Figure 1) was fabricated and packed with silica-coated paramagnetic particles (MagneSil™)
(Figure 1 inset) as the solid phase. With this device, a 50-fold volume reduction and 15-fold concentration enhancement
Inlet
Weir
Outlet
Figure 1: vrSPE device (1 cm to weir, 1 mm line width, 200 µm deep, 20 µm weir depth) packed with MagneSil™ solid
phase. SEM image of MagneSil™ particles is shown enlarged [4].
is achieved [5]. Additionally, an increased volumetric throughput is achieved due to the increase in channel width (2.6fold) in comparison to other µSPE devices. Since a wide range of large volume sample types exist in the forensic sector,
‘mock’ samples were prepared to demonstrate the wide spread application of the vrSPE method. The extraction process
using the device consisted of three steps: sample load (following conditioning of the silica solid phase with 6 M GuHCl,
pH 6.1), wash with isopropyl alcohol (to remove cellular and extracellular material), and elution of the DNA in low ionic
strength buffer (10 mM Tris pH 8). Each step was completed at a flow rate of 15 µL/min. A blood sample containing
140 nL of whole blood was prepared in 6 M GuHCl, pH 6.1 with proteinase K and DNA was extracted from the sample
using vrSPE to determine the effectiveness of the method for application to low template samples. Both blood and semen stain samples on cotton were also prepared and exposed to 56 min of UV light (equates to 4 months and 8 days outdoors) to simulate exposure to sunlight. The DNA was purified from both samples using the vrSPE method. Lastly,
DNA from a bone sample (obtained from the Armed Forces DNA Identification Laboratory) that had undergone demin978-0-9798064-3-8/µTAS 2010/$20©2010 CBMS
770
14th International Conference on
Miniaturized Systems for Chemistry and Life Sciences
3 - 7 October 2010, Groningen, The Netherlands
eralization and concentration [3] was extracted using the vrSPE method. After purification using vrSPE, all samples collected during the elution step were subjected to amplification with a commercially available forensic STR (short tandem
repeat) amplification kit, used for human identification, followed by separation/detection using an ABI 310 Genetic Analyzer.
RESULTS AND DISCUSSION
The microfluidic vrSPE method developed in this work decreases analysis time for large volume sample processing
when compared to conventional purification approaches and chip-based µSPE (due to the decreased flow rate used for µSPE
in comparison to vrSPE). The method also utilizes a high DNA binding capacity phase, 50.3 (± 5.6) ng DNA/µg particles
[5], ensuring DNA will not be lost to the competitive binding of other analytes in the cellular debris or to contaminants when
biological samples are purified. Additionally, the closed environment provided by the microdevice makes the method ideal
for forensic analysis, as reduction in the number of sample transfer steps (common to conventional analysis) decreases the
possible points of entrance for contaminants or DNases – a top priority when working with samples that may be degraded or
compromised.
D3S1358
Amelogenin
D7S820
TPOX
CSF1PO
TH01
D16S539
Figure 3: Full STR profile resulting from amplification using the COfiler® amplification kit of DNA purified from a large
volume sample (500 µL) containing 140 nL whole blood using the vrSPE method.
To test the utility of the vrSPE method for application to samples that contain a low concentration of DNA (low DNA
template), DNA extraction from a large volume sample (500 µL) containing 140 nL of whole blood, which equates to a
1:3,500 dilution of whole blood, was attempted using the vrSPE method. The extracted DNA was then amplified using a
commercially available STR (short tandem repeat) amplification kit. Short tandem repeats are typically tetranucleotide repeats present in regions known as loci that are found on chromosomes within the human genome. The number of repeats at
each of these loci varies from person to person providing a highly discriminatory tool for human identification. After amplification and separation and detection a full STR profile (7 of 7 loci present) was obtained (Figure 2). This demonstrates that
even at low quantities of template in a large volume sample that the vrSPE method can be used for successful purification.
These provide significant improvement over extractions reported previously using the same solid phase, but in an automated
robotic platform for large volume extraction [6] where locus dropout began to occur at a 1:1,000 dilution of blood. Not only
are large volume, low DNA concentration samples common, but samples that require solubilization from a surface that has
been exposed to harsh environmental factors are also encountered. To determine whether the vrSPE method could be used
for DNA purification from this sample type, DNA from blood-stained cotton that had been exposed to UV light (equivalent
to 4 months and 8 days outdoors) was purified using the vrSPE method, amplified using a commercial STR PCR kit, and
resulted in a full STR profile (Figure 3A) (16 of 16 loci present). Although UV exposure causes DNA damage [7], the
DNA is not completely degraded and DNA could still be successfully extracted by the vrSPE method, demonstrating the
broad spectrum of samples the method is capable of processing. The method was then applied to a semen stain on cotton
that had also been exposed to UV light (equivalent of 4 months and 8 days outdoors). Following purification with vrSPE,
amplification with a commercial STR PCR kit, and separation and detection, a full STR profile (16 of 16 loci present) was
obtained (Figure 3B). This shows that DNA from environmentally-degraded samples can be successfully purified using the
vrSPE method.
D8S1179
A
D21S11
D7S820
D13S317
D3S1358
D16S639
TH01
D19S433
VWA
TPOX
D5S818
Amelogenin
771
FGA
D18S51
CSF1PO
D2S1338
B
Figure 3: Full STR profile resulting from amplification using the Identifiler® amplification kit of DNA purified from a UV
exposed blood stain (A) and semen stain (B) using the vrSPE method.
To further test the method, DNA from a bone sample that had been subjected to demineralization and concentration was
purified using the vrSPE method. Following amplification, a STR profile, with 15/16 alleles present, resulted (Figure 4).
These results demonstrate the first development towards a microfluidic DNA purification method for bone samples.
Figure 4: STR profile (15/16 alleles present) resulting from amplification using the MiniFiler™ amplification kit of DNA
purified from a bone sample.
CONCLUSION
The work shown represents the first illustration of a microchip-based large volume DNA purification method capable
of handling low template samples (generated by dilution), environmentally-degraded (by UV-induced degradation) biological stains, and bone. The vrSPE method is advantageous for the forensic community as it provides a reduced analysis time, a closed environment to reduce contamination, and has been shown successful for DNA purification from samples that are commonly encountered in forensic casework analysis.
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