| 1. |
- Evander, Mikael, et al.
(författare)
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Acoustic Differential Extraction - ultrasonic DNA-extraction from sexual assault evidence
- 2007
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Ingår i: International Congress on Ultrasonics. ; 1, s. 151-151
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Konferensbidrag (refereegranskat)abstract
- Isolation of the male and female DNA is one of the most important steps in obtaining the DNA profile of the perpetrator in sexual assault cases. The sample is obtained by taking a vaginal swab containing both epithelial cells from the female and sperm cells from the male from the victim. The purity of the extracted male fraction decides whether or not a single-source DNA profile of the suspect will be obtained or not. The existing techniques are have poor separation efficiency, time-consuming, labour-intensive and are neither easily automated nor integrated with further analysis steps. A novel method of DNA extraction based on ultrasonic trapping, Acoustic Differential Extraction, has been developed. A microfluidic device using laminar flow valving and miniature PZT transducers retains the sperm cells while mobilizing the female fraction into a separate outlet. The device was evaluated using a mock sexual assault sample and the separated fractions were analyzed using quantitative PCR and STR-profiling. A 16-fold enrichment of the male fraction, making an originally hard-to-detect-male DNA profile readily profiled, has been demonstrated. The STR-profiling of the male and female fractions showed a male fraction purity of up to 99 % making it possible to obtain a single-source DNA-profile of the suspect. The microfluidic format of the device makes it possible to downscale the sample time from 4-8 hours to 10 minutes. It is also possible to integrate this method with further downstream analysis steps necessarey for the full forensic DNA analysis.
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| 2. |
- Evander, Mikael, et al.
(författare)
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Using Acoustic Differential Extraction to enhance analysis of sexual assualt evidence on a valveless glass microdevice
- 2006
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Ingår i: Proceedings of µTAS 2006 Conference. ; 2, s. 1055-1057
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Konferensbidrag (refereegranskat)abstract
- The isolation of male and female DNA is an important step in the analysis of sexual assault evidence. A vaginal swab with female epithelial cells and male sperm cells is obtained from the female, and it is vital to separate the male and female fractions in order to obtain a single-source DNA profile of the suspect. In the case of a low abundance of sperm cells, it is very important that no cells are lost in the isolation step. The conventional isolation method used in the forensic DNA laboratories, differential extraction, is a time-consuming step, requiring up to 24 hours. It is neither highly amenable to automation, nor can it be easily integrated with other steps of the analysis. Therefore, a novel method of performing the isolation of male and female fractions of biological material from sexual assault evidence has been developed, termed acoustic differential extraction (ADE). After selectively lysing the female epithelial cells while keeping the sperm cells intact, the sample, now containing sperm cells and female cell lysate, is infused in a 900 μm wide and 70 μm deep microfabricated glass channel with miniature piezoelectric transducers mounted at the bottom of the channel, as shown in Figure 1. Upon activation of the ultrasound, the sperm cells will be trapped in a standing wave1 while free DNA will not be retained. The sperm cells, levitated in the 3D fluidic space above the transducer, can be washed with buffer and the unretained biological material directed to an output reservoir. Using laminar flow valving2, the sperm cells can be released and directed into a separate output reservoir in anticipation of DNA analysis, see Figure 2. With the purpose of evaluating the ADE microdevice for the collection of the two output fractions (male and female), preliminary work used a mock sexual assault sample created with polystyrene microparticles as sperm cells and Evan's Blue dye as female cell lysate. The particles were trapped over the transducer and the dye was directed to the female outlet reservoir as shown in Figure 3. After washing the particles, the ultrasound was deactivated and the flow redirected in order to collect the particles in the male outlet reservoir. A biological sample consisting of sperm cells and lysed female epithelial cells was subsequently tested by infusion into the device for five minutes while trapping the sperm cells over the transducer (Figure 4). The trapped sperm cells were washed with PBS for five minutes, then released and collected for analysis off-chip. DNA from the isolated cells was extracted with a commercial DNA extraction kit and analyzed with a duplex quantitative PCR assay3 to show the sample purity. An example of the qPCR data obtained is provided in Table 1. The results show that a highly-enriched sperm cell fraction can be obtained with the ADE technique. It is reasonable to expect that this technique can be integrated with on-chip downstream sample processing, e.g. DNA extraction and amplification. This would greatly diminish the analysis time from 24 hours to approximately 60 minutes. The time savings, in combination with the possibility to create a fully automated system, gives the ADE technique the potential to significantly alter the means by which sexual assault evidence is processed in crime laboratories today.
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| 3. |
- Norris, Jessica Voorhees, et al.
(författare)
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Acoustic differential extraction for forensic analysis of sexual assault evidence.
- 2009
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 81:15, s. 6089-6095
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Tidskriftsartikel (refereegranskat)abstract
- Forensic DNA analysis of samples obtained from sexual assault evidence relies on separation of male and female components of the recovered genetic material. The conventional separation method used by crime laboratories, differential extraction (DE), is one of the most time-consuming sample preparation steps, requires extensive sample handling, is difficult to automate, and often results in inefficient separation of female DNA from the male sample components. To circumvent conventional DE, acoustic differential extraction (ADE) analysis was developed on a microfluidic device. The ADE method relies on acoustic trapping of sperm cells in the presence of epithelial cell lysate (which is unretained), and laminar flow valving to direct the male and female fractions to separate outlets. Following the separation of sperm from epithelial cell lysate, DNA extraction, quantitation, amplification, and separation were performed using conventional laboratory methods. The results show that highly purified male and female fractions can be obtained with the ADE microdevice from mock sexual assault samples in 14 min. ADE analysis provides the potential to significantly alter the means by which sexual assault evidence is processed in crime laboratories.
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