Developing microchips for DNA analysis
Enabling rapid DNA analysis at a crime scene
Yvette van ‘t Zand (12743232) Master Forensic Science December 11, 2020 Supervisor: Dr. Brigitte Bruijns Examiner: prof. Dr. Ate Kloosterman 6371 words
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Table of content
Abstract p.3
List of abbreviations p.3
1. Introduction to microfluidic DNA analysis p.4
2. Quality vs. Speed p.5
2.1. Sensitivity 2.2. Speed
3. Common LOC designs and performance p.6
3.1.Sample input 3.2. Quantification 3.3. Amplification 3.3.1. Cycling designs 3.3.2. PCR parameters 3.4 Detection
4. The effect of chip materials p.10
4.1. Surface inhibition 4.2. Silicon, silica or glass 4.3. Polymers
5. The implementation of rapid DNA technology in the Netherlands p.12
6. Discussion p.16
6.1. Challenges remaining 6.2. Other developments
6.3. The direction of future research
7. Bibliography p.18
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Abstract
Fast DNA analysis is now becoming available for forensic practice with the development of microfluidic devices. These devices can be taken to the crime scene and incorporate all laboratory steps of DNA analysis within a single portable system. Microfluidic DNA devices have become very fast and can generate a DNA profile within 2 hours, they are also becoming increasingly sensitive, but for most crime scene samples, the sensitivity is still lower than the sensitivity that can be obtained in the laboratory. This thesis explores different designs of chips and discusses several parameters that can be optimized to increase speed and sensitivity. Several adjustments can be made to increase the sensitivity, like increasing the DNA concentration in a sample or by using a droplet-based approach. Another option is to only use high quality samples in a rapid analysis like saliva or blood traces. Finally, the thesis explores how these devices are currently implemented in Dutch forensic practice and what additional research is required for further development and improvement of mobile DNA analysis.
List of abbreviations
COC Cyclic olefin copolymer
DNA Deoxyribonucleic acid
ENFSI European Network of Forensic Science Institutes
LOC Laboratory-on-a-chip
LCN Low Copy Number
NFI Netherlands Forensic Institute PDMS Polydimethylsiloxane
PCR Polymerase Chain Reaction PMMA Polymethylmethacrylate
qPCR quantitative PCR
SOCO Scene of Crime Officer SWGDAM
Scientific Working Goup on DNA Analysis Methods
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1. Introduction to microfluidic DNA analysis
The development of rapid, mobile DNA analysis technologies is slowly revolutionizing forensic investigations. These DNA techniques incorporate all laboratory steps on a microscale using microfluidics. A lab-on-a-chip (LOC) therefore offers the advantages to analyze samples rapidly at the crime scene to identify and eliminate suspects within the first, most crucial, hours of an investigation (1).
In the laboratory, forensic DNA samples are analyzed in five consecutive steps: sampling, sample work-up, quantification, amplification and detection (fig.1)(2). A lot of research has already been performed to develop and validate rapid methods for direct implementation in forensic practice (3–8). Different chip designs have been developed and optimized to fully integrate all laboratory steps to create sample-in, answer-out systems (9–14), like the RapidHIT 200 (IntegenX) (15–17), and the ANDETM (18–22) systems.
Current platforms can yield full DNA profiles in less than 2 hours, which is much faster than the fastest laboratory track at the Netherlands Forensic Institute (NFI), taking 6 hours (23). Another advantage that DNA analysis LOCs offer is their mobility. The devices are designed to have a small footprint and can therefore be taken to a crime scene easily and operated by a trained Scene of Crime Officer (SOCO). Finally, the methods generally use less reagent than the laboratory approach, making LOCs cost-effective. All steps ideally take place within a closed environment that is disposable, ensuring the chain of custody and minimizing contamination (5,7).
Although reference samples can already be analyzed with a good success rate, the analysis of crime scene samples remains challenging (3,18,24). Crime samples are much more variable than reference samples, since they originate from different cell types and surfaces and are exposed to variable environmental conditions. These factors affect DNA concentration and quality, resulting in different success rates for different trace DNA samples (25). In addition, crime samples can contain several types of inhibitory substances like humic acid or urea, which interfere with the analysis (26,27). This requires LOCs for use at the crime scene to be able to process different concentrations of DNA and to be resistant to common inhibitors, while remaining faster than laboratory techniques and maintaining profile quality.
This thesis will review the current status of microfluidic devices for DNA analysis at the crime scene and discuss the advantages and challenges of the different technologies. Finally, the future perspectives of mobile DNA technologies will be outlined with a special focus on the implementation of the RapidHIT system in Dutch forensic casework.
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2. Quality vs. Speed
As microfluidic devices for forensic DNA analysis become faster, the quality of the resulting profiles should remain high. As forensic samples can be highly variable in nature with different concentrations, sample qualities or mixture ratios (25), the LOCs would ideally be able to process a wide variety of samples within a small amount of time while maintaining quality. However, in design, increasing sensitivity (i.e. the ability to process trace amounts or contaminated DNA) often detracts from speed and vice versa. This chapter explores why the dilemma of speed and quality is important and its implications for chip design.
2.1. Sensitivity
The quality of an output DNA profile depends on the ability of a LOC to process a certain sample. As samples often differ, it is advised to test the limits of detection by assessing the performance of the LOC with variable sample input during developmental validation. The input should vary in both quantity and quality (3,28). This ensures that the challenges of analyzing case samples, like contamination and varying concentrations are properly addressed. However, it has been shown that reference samples, like buccal swabs, can be processed on a rapid device to produce DNA profiles with a sufficient quality, but the processing of crime samples is still under development (3,24).
Mapes et al. have shown that the success rate of laboratory and microfluidic DNA analysis depends on the DNA concentration in the samples, as well as on the type of exhibit the sample was taken from (29,30). Recently, some adjustments to increase the success rates of microfluidic analysis of low DNA content samples have been presented, like the optimization of sample work-up for increasing the DNA concentration (13) or droplet-based chip design, which has a resolution of up to a single cell and enables removal of inhibitors (12,26). The latter, however currently takes up to 22 hours of processing and is therefore not (yet) fast enough to apply in forensic practice. The different chip designs will be discussed in more detail in chapter 3.
2.2. Speed
Currently the fastest DNA track in the Netherlands is performed at the NFI and takes six hours (23). Ideally, the DNA analysis on a LOC is even faster enabling DNA analysis at the crime scene during the investigation. Faster results can guide SOCOs within the first ‘golden’ hours of the investigation and might lead to quicker apprehension of potential suspects. Generally, DNA analysis on a chip can be faster than laboratory analysis, because the thermal inertia of small devices is low. This results in faster thermal cycling with the same effect (16). The speed can further be increased by adjusting PCR parameters, through improving cycling design and by optimizing STR kits. As PCR is the most rate-limiting step in DNA analysis, this thesis mainly focuses on on-chip PCR design as a point for development.
The following chapters will focus on different chip designs and their performance with regards to speed and sensitivity, followed by a critical discussion about the performance of these devices in forensic practice and improvements that are still required.
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3. Common LOC designs and performance
A wide variety of microfluidic devices has been developed over the past decade. Some of them integrate all laboratory steps: sample work-up, amplification, quantification and detection (fig.1) on-chip, while others can only perform amplification and/or detection and require the input of purified DNA. The different designs have their own advantages and disadvantages and affect the performance of a device. This chapter reviews existing LOC designs for every step of the analysis process, but with a special focus on PCR technology.
3.1. Sample input
In 2016, Mapes et al. demonstrated that there is a relationship between the success rates of rapid DNA analyses and the DNA concentration and the exhibit that the sample was recovered from. Balaclavas, blood and cigarette butts showed good success rates and a high concentration of DNA, whereas items like keys, tools and cartridges showed a low concentration of DNA and lower success rates (29,30). This research shows that it is important to consider what sample inputs can be analyzed in a mobile DNA analysis system.
Some systems require the input of purified DNA and will directly start the amplification reaction. The downside is that the sample must first be processed in the laboratory before it can be inserted into a microfluidic device (14,16), which makes the process more time-consuming and increases the risk of contamination. Other systems require the input of a swab and have an integrated module for cell lysis and DNA extraction and purification. The ANDETM chips use a silica-based
extraction method with an ethanol-based wash to prepare the DNA for amplification (13). Cell lysis and DNA extraction can also be performed in droplet-microfluidics, in which the DNA is trapped in agarose droplets, and the cell lysate can be washed out, while the DNA remains trapped (12).
ANDETM recently developed a new type of system that is specifically designed to analyze low-level
DNA samples. In order to increase the success rate, the manufacturers developed a special module that improves the DNA concentration after purification (fig.2). The sample flows through the module and encounters a filter that traps DNA molecules but excess solvent flows out into a waste chamber. The final concentration is thereby increased, but depends on the recovery efficiency (31).
Figure 2: ANDETM solution for increasing DNA concentration. The sample flows in at (1), then the sample gets separated into excess eluate that flows to the waste chamber through (3) and concentrated DNA that flows through (2) for further processing. Source: Tan et al. (2013) (13).
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3.2. Quantification
In the laboratory, samples are quantified before amplification to ensure that the amplification reaction is most efficient and to prevent having too much DNA in the detection phase, since that would distort the fluorescent signal in the profiles (2). In fully integrated systems like ANDETM,
the correct DNA quantity can be obtained through the use of silica filters (13). It is also possible to apply quantitative PCR (qPCR) to monitor the amount of DNA in the sample (32). This has not yet been incorporated in devices that are currently implemented, but in the future the use of qPCR on-chip has the potential to improve the efficiency of microfluidic DNA profiling.
3.3. Amplification
There are several ways to set up a PCR amplification on-chip. By altering the cycling design, the speed of the amplification can be increased, but it can also determine how easily chips can be manufactured and how easily the amplification can be integrated with other analysis steps. Additionally the sensitivity and speed are strongly affected by cycling design, e.g. small amounts of DNA require more thermal cycling, but that also requires more time. Also PCR parameters like cycling time, temperature, reagents etc. need to be fine-tuned to optimize the reaction.
3.3.1. Cycling designs
PCR can be performed in a well-based design, where the sample and reagents are loaded into a reaction chamber that is subsequently heated and cooled. Alternatively PCR can be performed in a continuous flow design, where the sample and reagents flow through thermally different areas on the chip. Finally, a relatively new design is the droplet-based approach, which provides some specific advantages and disadvantages.
Well-based
In a well-based amplification system, the sample and reagents are loaded into a well or chamber, which is subsequently heated and cooled (5). Overall, microfluidic devices that are used for PCR have the advantage that they have a low thermal mass and low thermal inertia, resulting in faster heating and cooling rates (6). In this approach, the chip needs to be heated and cooled in its entirety, thereby showing a higher degree of thermal inertia than continuous flow devices. However, regarding the size of the chip and volume of reagents, the effect of the inertia on the speed is negligible.
Continuous flow
In contrast to a well-based approach, the sample in a continuous flow device is not stationary, but rather moves through microfluidic channels. There are several types of continuous flow chips, which can be subdivided in the fixed-loop, closed-loop (5). In both fixed-loop and closed loop systems, the sample moves through fixed temperature zones. The main difference between the two systems is that in fixed-loop designs, the number of cycles is predetermined by the number of meanders in the chip microchannels, whereas the number of cycles in a closed loop device can be manipulated (5,6). For crime samples, it can be a big advantage to use a closed loop chip, because different numbers of cycles can be used to analyze different DNA concentrations.
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Droplet-based
In a droplet-based approach, small droplets are added in a medium (e.g. aqueous droplets in a hydrophobic medium), in which each droplet acts as a single reactor (5). A droplet-based PCR chip design can be either stationary or be in a continuous-flow, depending on the application (4). In the stationary design, the droplets van be closely monitored and manipulated (33). The great advantage is that a droplet-based approach can provide a high degree of resolution up to the level of a single DNA molecule, which is helpful for crime samples with little template DNA. Geng et al. designed a droplet-based chip (fig.3) which can perform STR multiplexing at a single-cell resolution. In their approach, DNA can be extracted and amplified within agarose droplets in an aqueous medium (12,26).
The speed of the PCR in droplet microfluidics is not necessarily faster than a laboratory procedure. The device designed by Geng et al. requires two rounds of PCR with 55 cycles in total, resulting in a 22 hour process (26). On the other hand, their approach is very sensitive and robust and offers the opportunity to wash away the most important contaminants that have an inhibitory effect by trapping the DNA molecules in the agarose, while smaller molecules are washed out (26). Finally, there is another advantage of droplet-based techniques relative to other cycling designs, since in other designs the sample and reagents can interact with the surfaces of the microchannel wall, thereby inhibiting PCR (34,35). In a droplet-based approach, the droplets generally do not contact the surface of the flow channels, resulting in a minimal PCR inhibition through this interaction (36).
Figure 3: Example of a droplet-based workflow. Agarose droplets are generated in an oil matrix. They can take up a cell and contain a primer bead with PCR primers attached to it. The DNA is extracted within the droplet and the lysate is washed away. The DNA is then amplified on the primer bead. Finally, a second round of bulk PCR is performed, after which the amplified material can be used for fragment analysis. Source: Geng et al. 2014 (12).
3.3.2. PCR parameters
Apart from choosing the right cycling design and chip material (chapter 4), it is also important to fine-tune the PCR parameters in each device. Changing the amplification parameters affects the speed and sensitivity as well as the efficacy of the reaction. Cycling parameters are: number of cycles, temperature and time of each step and the reaction components that are used. Specific PCR conditions like cycling times and the number of cycles are often optimized to fit the type of chip and the STR kit that is used. In addition, the cycles itself can be shortened by combining the extension and annealing steps.
Page | 9 Table 1 summarizes the PCR parameters for three different, but similar fully integrated devices: The commercially available ANDETM and RapidHIT systems and a developmental device by
Cornelis et al. (10). All devices are well-based and use thermal cycling, but each system has its own characteristics, since they were optimized for different PCR kits. The success rates for the ANDETM and RapidHIT systems are good, but these were obtained with samples that have a high
concentration of DNA. The system by Cornelis et al. seems to be the most sensitive and fast system, but has not yet been validated to the extent of the ANDETM and RapidHIT devices.
Table 1: Comparison between parameters and performance of different rapid DNA analysis systems. The ANDETM and rapidHIT systems are compared as well as a developmental system by Cornelis et al.
3.4. Detection
In order to obtain an STR profile, capillary electrophoresis of DNA fragments with the detection of fluorescently labeled probes is used both on- and off-chip. The PCR kit contains fluorescently labeled primers to label all STR loci for detection. After amplification, the sample can be mixed with electrophoresis buffer and subsequently separated by size in a gel matrix. Finally the probes are detected and a profile can be generated by integrated expert software (2,5,13)
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4. The effect of chip materials
There are many different types of chip materials that can be used in a microfluidic design. Some chips use polymers, others use silicon or glass, all with their own characteristics. Some demands the material will have to fulfill for on-chip PCR are thermal conductivity and a low inhibitory effect. For fluorescent detection the material should be transparent in a wide optical range and have low autofluorescence (5,37).
4.1. Surface inhibition
The inhibitory effect of some materials has been studied extensively over the past decade, showing that many chip materials show some adsorption of PCR components like template DNA, primers or Taq polymerase (34,35). Also, the extent to which PCR is inhibited depends on the contact between the channel surface and the sample/reagent mixture. Some factors determining this contact are the surface-to-volume ratio (SVR), the exposure time, the flow velocity and the length of the channels (34). However, only an increased channel length (34) and a bigger SVR (35,37) have been shown to be inhibitory.
By using a droplet-based design (chapter 3), the inhibitory effect of the channel material is minimal since the SVR is negligible as the droplets do not come into contact with the surface (36). On the other hand, the surface does have to be compatible with the carrier fluid that carries the droplets through the channels.
4.2. Silicon, silica or glass
In other designs, both well-based and continuous flow designs, the sample and reagents do come into contact with the channel surface, although in differing degrees of exposure. The biggest inhibitory effect of the surface to the PCR reaction has been shown in silicon channels, since surface silanol (Si-OH) easily reacts with polar molecules like DNA and polymerase (37). This interaction can be prevented by passivation of the surface through using silica (SiO2) or special
coatings, like silica or SF6 or C4F8 plasma coatings (35). Aside from coatings, active competition
can also be used to minimize material inhibition. For this, bovine serum albumin (BSA) can be used, which can bind to the surface and therefor occupy the channel wall. Mixed results have been published leaving it unclear to which extent BSA minimizes PCR inhibition (35,37,38). Despite the inhibitory effect that silicon shows, it does have a great thermal conductivity making it well suited for thermal cycling (10).
Using silica or glass as surface materials provides some advantages since they are smooth and compatible with the sample and reagents. They are also better suited for integration with electrophoresis and detection, because they have a low electrical conductance, low autofluorescence and are, unlike silicon, transparent in a broad optical range (35). However, glass must be coated before it can be used to prevent reaction inhibition (38).
4.3. Polymers
Another option is the use of polymers, which come in various alternatives like cyclic olefin copolymer (COC), polymethylmethacrylate (PMMA) and polydimethylsiloxane (PDMS). All materials have different properties and vary in reaction compatibility, transparency and autofluorescence. The general benefit of using polymers for chip fabrication is that they are generally cheaper and therefore more suited as a disposable material, which is convenient in forensic practice to prevent contamination (14).
Page | 11 COC is a very robust material due to low surface absorption and a good compatibility with acids, alkalines and polar solvents (38). The material would be particularly useful for devices that integrate a fluorescence-based detection step, because it is transparent in a broad optical spectrum and shows very little autofluorescence (fig.4) (13).
Figure 5: Autofluorescence of glass and cyclic olefin copolymer with different thickness in the visible spectrum. COC shows lower autofluorescence than glass, and one emission peak. Source: Tan et al. 2013 (13).
PMMA and PDMS can be used for droplet-based PCR, since the materials show good light transmittance and compatible with the reagents. The hydrophobic surface of PDMS makes it well-suited for water-in-oil droplets. However, the use of both PMMA and PDMS should perhaps be restricted to droplet-based microfluidics, since there are strong signs of PCR inhibition when there is a big SVR (35,37). The level of inhibition by PDMS is lower than the level of inhibition by PMMA (37). The inhibition can seemingly be reduced through the use of BSA (5,37). Finally, the thermal conductivity of PMMA and PDMS is relatively low (39,40), which makes the materials not very well suited for well-based PCR, but beneficial for continuous flow chip, since a low thermal conductivity is required to maintain fixed temperature zones (41).
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5. The implementation of rapid DNA technology in the Netherlands
In November this year, the Dutch law for DNA analysis in criminal cases was changed in order to allow for the use of mobile, rapid DNA analysis technologies on the crime scene. With that, Dutch law enforcement can now start the implementation of rapid DNA technologies in forensic
practice. The stepwise research into the deployment of the RapidHIT200 technique is led by the Amsterdam University of Applied Sciences (42). This chapter will evaluate the RapidHIT200 technology and its performance. Then, the process of implementation will be discussed.
5.1. The RapidHIT technology
Several RapidHIT systems are on the market, each with their own advantages and disadvantages. The first one that was developed is the RapidHIT200 (IntegenX), which is optimized to process buccal swab samples, which are assumed to contain high quality and high quantity DNA of a single donor. The system performs all steps, i.e. cell lysis, DNA isolation, STR amplification, electrophoretic separation, fluorescent detection and data analysis, in under 2 hours. It can process five samples and three control samples at the same time (16). The
downside of this procedure is that it is not possible to analyze only one sample at a time, so the RapidHIT ID (fig.6) was developed, which can analyze a single sample (43). The RapidHIT ID is made specifically for the use in decentralized environments like a crime scene and uses low cost disposable cartridges. It is also designed for the processing of single source samples (15). The processing time is reduced to 90 minutes (15,43).
Figure 4: RapidHIT ID system (IntegenX). The left image shows a photograph of the desktop system and the insertion of the disposable cartridge. Figure A shows the disposable cartridge in which the swab can be inserted and which performs cell lysis and amplification. Figure B shows the primary cartridge which performs the capillary electrophoresis and fluorescence detection steps. Source: Salceda et al. 2017 (15).
The systems were originally designed to be integrated with the PowerPlex16 STR kit (Promega), but a desire to analyze more loci for international communication and reduction of adventitious matches resulted in the use of different STR kits like the GlobalFiler Express Kit (ThermoFisher Scientific), which is optimized for 24 loci, of which 21 are autosomal and 3 are sex-specific (15,44).
Cell lysis is performed through chemical lysis and solid phase DNA isolation. The amplification is then performed in a single PCR chamber and therefore follows a well-based design. The
amplified product is subsequently separated through electrophoresis and can be detected in the primary cartridge. The RapidHIT systems have integrated software for data analysis to generate output profiles (44).
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5.2. Performance
The performance of rapid DNA analysis devices is generally tested according to the SWGDAM validation guidelines. For STR-based analysis systems, the main points that have to be tested are sensitivity, stability, precision, repeatability and reproducibility. The sensitivity reflects the minimum amount of input that is required to obtain full profiles, while the stability shows the compatibility of the system with different types of samples and surfaces. The precision can be measured by comparing the alleles that were called with the allelic ladder to evaluate the base pair resolution. Finally, the assay has to be human-specific and should be tested for its
recognition of mixtures.
The RapidHIT system has mainly been validated and optimized using the GlobalFiler Express Kit. The optimal parameters are shown in figure 7. The figure shows that there are no significant differences in profile peak height or heterozygote ratio when changing PCR parameters. It does reflect that cycling parameters may be slightly optimized (44).
Figure 5: Changing cycling parameters and the effect on profile peak height and heterozygote peak ratio. Asterisks indicate the recommended parameters. The graph shows that changing the temperature of PCR steps by two degrees or manipulating the final extension time by 4 minutes has no significant effect on the peak height (blue) or the heterozygote peak ratio (green). Increasing the number of cycles to 29, might however have a positive effect on the peak height, although this is not significant. Source: Hennessy et al. 2014 (43).
Validation studies also showed that common oral inhibitors (e.g. tobacco or coffee) did not affect the results. A precision test revealed that the peaks show a 0.5 basepair resolution. Additionally, no cross-contamination was found between or within runs. Finally mixtures up to a mixture ratio of 1:9 were correctly identified by the software, an example of which is shown in figure 8 (15,44). Finally, success rates can be reported as the percentage of full profiles that are
concordant when comparing the rapid method to the laboratory procedure. With high quality single-source samples the success rate had an average of 90% with a 60% lower limit (45).
Figure 6: Identification of minor alleles in a mixture with a ratio of 1:9. Asterisks indicate the minor alleles. Source: Hennessy et al. 2014 (43)
Page | 14 Except from one study that attempted an internal validation, but was interrupted by technical malfunctioning of the system (46), the validation studies show promising results. Nevertheless, the vast majority of research is performed with single source, high quality and high quantity samples like buccal swabs or blood samples, while in casework the quantity, quality and nature of samples greatly varies.
Only one report of the use of the RapidHIT technology in forensic casework has been published, showing how the standard RapidHIT protocol was used for 36 samples in 9 Singaporean cases. Blood, bone marrow, semen and cigarette butts were analyzed with the RapidHIT, the success rates1 of which are shown in table 2. 89.7% of alleles were called correctly, but the laboratory
procedure still strongly outperformed the rapid technology in identifying mixtures and sensitivity (47).
The same research group then performed a sensitivity study with different dilutions of blood, varying from 50 to 0.125 µL. The analysis of these dilutions on the RapidHIT system were also compared to the performance in the laboratory. The results of the success rates are shown in table 3. It is clear that there is a big sensitivity gap between the laboratory protocol and the rapid protocol, however the success rates of blood, bone marrow, fingernail clippings and cigarette butts could be considered sufficient in some high profile cases when weighing the speed against the sensitivity (48). A more graphical representation of the difference in sensitivity is added in the appendix.
Table 2: The success rates of the DNA analysis of different samples with the RapidHIT technology compared to the standard laboratory procedure. Success rates are defined as more than 50% and more than 80% of alleles called. Source: Thong et al. 2015 (46).
Table 2: Success rates for different dilutions of blood. The percentage of alleles calles as well as the profile qualities are compared for analysis on the RapidHIT system and according to the laboratory protocol. Source: Thong et al. 2015 (47).
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5.3. Implementation
Despite some challenges and a lot of uncertainty remaining, the technology is going to be implemented in Dutch Police units. Since 2017 the LocalDNA project has been running within the University of Applied Sciences in Amsterdam to investigate the challenges of rapid DNA analysis implementation together with other stakeholders (49). The operation of an analysis system by SOCOs required the law to be changed (50) and demands an elaborate communication system for close cooperation between Police, prosecution and forensic experts at the NFI (49). A change in law has been signed by the Dutch King in November of this year. Now, SOCOs are legally allowed to operate a rapid DNA system and to insert samples if requested by the public prosecutor. The results will be sent to a forensic DNA expert who can evaluate profiles and communicate the outcome to the public prosecutor prematurely (i.e. before submitting a formal report) (51). This aids in a quick identification of potential suspects and helps in scenario
building by the crime scene investigators (52–55). Finally, the new law requires the system to be correctly accredited2 (51).
Interestingly, a lot of effort of the localDNA project has led to the publication of papers regarding the cognitive aspect of crime scene investigation. The hypothesis is that new information, like identification of a person through rapid analysis of a trace, alters the decision-making behavior in crime scene investigators. The availability of additional information may lead to confirmation bias and tunnel vision (53,55). The research however, showed that the possibility to use rapid DNA techniques did not alter the number of traces that were collected and led to similar final scenarios. It also showed that investigators that used the rapid method arrived at more accurate scenarios, especially when multiple perpetrators were involved (53). Another study, in which some investigators received identification information early in the investigation and others received the information later in the investigation. Both groups arrived at the same scenario, but the early group did put more emphasis on the traces that provided a match (55). In conclusion, there is no clear effect of rapid DNA identification on CSI decision-making.
On the other hand, the decision whether or not a trace is suited for rapid DNA analysis should be guided. Some traces have a bigger probability of success on the rapid device than others and sometimes the use of rapid technology is considered destructive, so the trace cannot be re-analyzed in the laboratory. It is therefore important to make the right decision considering both the speed and sensitivity of analysis. Mapes et al. created a model to guide SOCOs in this
decision, taking into account the factors of success and time pressure. The model led to more transparent and deliberate decision making by SOCOs and could therefore aid them to choose suitable traces for rapid DNA analysis (56).
In the future, the performance of the rapid DNA analysis system in casework should be
evaluated, while in the mean time research is conducted to improve the sensitivity of devices.
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6. Discussion
The implementation of rapid DNA technologies that can be used at a crime scene is very promising and feels like the CSI series is becoming reality. The ability of SOCOs to obtain DNA profiles at a crime scene might provide early clues to the identity of potential suspects and the future will tell us how effective this will be. Looking at the future, there are still many challenges ahead, since the performance of LOCs for the analysis of trace samples should still be improved. Also, it is important to await studies that report on the performance of LOCs in forensic practice to see what the success rates are with real crime samples.
6.1. Challenges remaining
Microfluidic DNA analysis can offer great advantages when used on the crime scene with crime samples. The mobility and speed can guide investigators early in casework and may lead to a quick apprehension of suspects. Also, the devices are easy to use and can, when fully integrated, perform the entire laboratory procedure in a closed environment, minimizing the risk of
contamination.
It is however a big leap to go from reference samples to crime scene samples, since these are very different in nature. Reference samples are (usually) a single person profile with a high quantity and quality of DNA, whereas crime samples come in many more varieties, as the quantity and quality can strongly differ. Additionally, crime samples are more likely to be mixtures and can be contaminated with PCR inhibitors like humic acid and urea (27).
Ideally, microdevices for forensic DNA analysis should be able to process many different types of samples while being as successful as the laboratory procedure, since it would be pitiful when a sample yields no profile on a microdevice, when it would have led to a usable profile in the laboratory. As mobile DNA technologies could soon be implemented in forensic practice, it is important to evaluate what challenges are still ahead and at what points LOCs can be improved to fulfill the requirements for implementation.
6.1.1. Requirements
To safeguard the quality of forensic microdevices, the European Network of Forensic Science Institutes (ENFSI) and the Scientific Working Group on DNA Analysis Methods (SWGDAM) set up validation guidelines to ensure LOC performance (3,28). The guidelines ensure that LOCs are able to handle different amounts of input DNA, that the results are robust and reproducible and that there is a high degree of concordance with other methods. Also, the chips need to
implement positive and negative controls and should be able to withstand some specific inhibitors (3,28).
Naturally, it is required that a rapid DNA technology is faster than the laboratory procedure, since the main advantage of these mobile devices is that they should give fast results. Otherwise, the quality of output profiles should be as close as possible to the standards that can be obtained in the laboratory. This requires LOCs to be very sensitive so they can process trace amounts of DNA that can be of poor quality and contaminated. The most sensitive microdevice is a droplet-based approach created by Geng et al. It is very robust and can generate profiles at a single cell level. However, this high resolution strongly detracts from speed, since the analysis takes around 22 hours (12,26).
Page | 17 The sensitivity can also be increased in other ways, as discussed in chapter 3, the ANDETM
system has developed a specific extraction method to increase the DNA concentration in the sample, thereby increasing the first pass success rates for low-level DNA samples (31). Also, adjusting PCR parameters, like different STR kits, different DNA polymerases and varying cycling times can increase the sensitivity of a LOC.
Lastly there is another way to approach this problem. Mapes et al. showed that the success rate strongly depends on the type of trace and the material that the trace is recovered from (29,30). This has been confirmed in validation studies (18). Therefore it might be interesting to look at traces that are most likely to yield full profiles on a microdevice, advice SOCOs to only select those for analysis. Traces that have a smaller likelihood of giving full profiles, should still be sent to the laboratory for analysis. In this way, some traces can still be analyzed quickly and reliably, while not wasting traces that are unlikely to be properly analyzed on a microscale.
6.2. The direction of future research
First of all, it is important to see how the rapid devices operate and perform in actual casework. So it is recommended to study the success rates of the devices as well as the experiences of SOCOs with them to identify factors that can be improved upon. Also, the validation reports of existing systems mainly communicated the minimal amount of DNA input for a full profile, while it might be interesting to see what the success rates are for partial profiles that are studied by a forensic expert after analysis.
In addition, the challenges with sensitivity have to be addressed. This can be approached by improving droplet-based designs so they become faster. The sensitivity and robustness of this approach is very promising, but has to become a lot faster, since 22 hours is still much slower than the fastest Dutch DNA track of six hours. On the other hand, it could also be very interesting to incorporate qPCR on the chip (32) to quantify the template DNA. With that knowledge, the STR parameters might ideally be adapted for each sample to obtain the most efficient results.
Improving the success rate of rapid DNA analysis can also be done by improving the concentration of DNA in a sample, for which a module like the filtration module in the ANDETM system (fig.2) is
promising. However, if the DNA concentration is low and the sensitivity of devices has reached its maximum, it is important to consider more research into the selection of traces as proposed by Mapes et al., who proposed a decision making model to guide SOCOs (56). This model could be tested in practice with the implementation of the RapidHIT to see how a model would fit within the work of SOCOs.
In conclusion, big steps have been made to make rapid DNA analysis at the crime scene possible, up to the point where the technology is being implemented in forensic practice. The current methods are very fast (<2 hours) compared to the laboratory process (6 hours), but not sensitive enough to process low template DNA samples. Some new technologies are being developed and existing methods are continuously improved to obtain a better sensitivity. For now, the devices can be used in actual casework to process high success-rate samples like blood and saliva which might potentially lead to quicker apprehension of suspects and better scenario formation by SOCOs.
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Appendix
1. Initial assignment
Forensic Expertise Area: DNA-analysis
DNA-analysis on a chip
SHORT DESCRIPTION
Unfortunately, biological traces found on the crime scene often contain a limited amount of
DNA. This while the DNA success rate is highly dependent on the DNA concentration [1]. A
lab-on-a-chip (LOC) or a microfluidic device (see some examples in the picture below) can be
described as a system in which multiple conventional lab techniques can be combined in one
device with a footprint of several square centimetres. These devices consist of enclosed
microchannels, whereby the changes of contamination are reduced. Other benefits of these
kind of systems are that less sample and reagents are required, shorter reactions times and the
small footprint make these devices suitable for use directly at the crime scene [2].
In this literature study we would like to investigate the possibilities of on-chip STR-PCR.
The following research questions need to be answered:
•
On-chip PCR:
o
Which cycling designs exist (e.g. well based)?
oWhat kind of chip materials are used?
o
What is used as input (e.g. lysate and how much)?
•On-chip STR-PCR:
o
What are the parameters used for STR-PCR (e.g. which kit, cycling times)?
oIs the device only used for PCR or are more functionalities integrated (e.g.
detection or sample prep)?
Analysis of specific (on-)chip aspects, like production methods, is not part of the literature
research. Micronit has enough experience on that aspect. Please focus on article published in
the last 5 years. Most interesting articles combine multiple steps of the DNA analysis process
and not only PCR.
REFERENCES
1. Mapes, A.A.; Kloosterman, A.D.; van Marion, V.; de Poot, C.J. Knowledge on DNA
Success Rates to Optimize the DNA Analysis Process: From Crime Scene to
Laboratory. J. Forensic Sci. 2016.
2. Bruijns, B.; van Asten, A.; Tiggelaar, R.; Gardeniers, H. Microfluidic devices for
forensic DNA analysis: A review. Biosensors 2016.
Page | 22
2. Search log
SEARCH LOG
Since I knew close to nothing about the subject at the beginning, I chose to start very
generally.
1. I took the papers that were given in the assignment and read them
top-to-bottom, then picked important papers from the reference lists. And read
some additional reviews.
2. I then searched for papers with microfluidics for DNA analysis in a
forensic context, mainly for on-chip amplification on Google Scholar and in
the UvA library. I filtered them to be published in 2016 or later to make
sure they were recent accounts
3. I added all the references I found to Mendeley and read all abstracts,
introductions and results.
4. Then I selected additional references from the reference lists of key
papers to get a more overall view.
5. For each chapter there are some keywords, which I searched in Mendeley
to retrieve the most important papers for that chapter.
6. The references for one chapter was categorized in tables, like the table
below, to make it easier to find them and summarize them so I could easily
incorporate them into the text.
7. Finally, if information was lacking I tried to find additional (often slightly
older) papers that could give a further explanation.
Polymer material Abbreviation References
Cyclic olefin copolymer COC (13,31,38,57)
polymethylmethacrylate PMMA (8,11,14,33,35,37–40)
SU-8 (5,37,38)
Norland Optical Adhesive 81 NOA81 (38,40) Polyethylene glycol diacrylate PEGDA (38)
