DNA Extraction and Quantification

DNA Extraction and Quantification A Alonso, Instituto Nacional de Toxicologı´a y Ciencias Forenses, Servicio de Biologı´a, Madrid, Spain ã 2013 Elsevier Ltd. All rights reserved.

DNA Extraction The process of isolating purified nuclear and/or mitochondrial DNA from both forensic specimens (blood, semen or saliva stains, hairs, muscle, bones, teeth, etc.) and reference samples (bucal swabs, blood spots on FTA, or liquid blood) is a crucial step to DNA profiling. Advances in forensic DNA extraction systems have been aimed at increasing the efficiency in the amount of purified DNA recovered (free from polymerase chain reaction (PCR) inhibitors) and automating the process for high-throughput analysis while maintaining a high integrity of the DNA molecule. Currently, the validated methods for DNA extraction most widely used in forensic laboratories can be classified into three groups on the basis of their purification strategies: organic (phenol–chloroform) extraction, solid-phase DNA extraction methods (silica based), and ionic chelating resins (Chelex). Specific procedures using some of these basic DNA isolation principles (or a combination of them) have been developed depending on the type of sample source. These include the differential lysis procedure for the selective extraction of sperm cells, special procedures for bone and teeth DNA extraction, the procedure for DNA purification on reference biological samples spotted on FTA paper, or previous selection of specific cell types by laser-capture microdissection coupled with DNA extraction. Automated DNA extraction procedures with different robotic platforms have also been implemented in forensic labs for high-throughput sample preparation, avoiding manual errors while improving sample tracking and reproducibility. Quality standards for DNA extraction in forensic labs include preventive measures against DNA contamination as well as the use of appropriate positive and negative controls for monitorization (Figure 1).

Organic (Phenol–Chloroform) Extraction Organic extraction has been one of the DNA extraction methods most used in the forensic field. The first step of any DNA extraction assay is the breakdown of cell membranes and proteolytic digestion in the presence of sodium dodecylsulfate (SDS), and proteinase K. DNA is first purified by mixing thoroughly the cell lysate with a phenol–chloroform solution followed by centrifugation in order to separate the organic phase, where proteins become trapped, from the supernatant aqueous phase, where DNA remains. DNA in the aqueous phase is further purified by precipitation with ethanol and finally resuspended in a low-salt buffer. For maximal DNA recovery and purity, the organic method protocol developed in many forensic labs involves a filtration purification step of the aqueous phase (instead of ethanol precipitation) using Centricon, Microcon, or, more recently, Amicon filter devices for DNA washing and concentration by

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centrifugation through membranes with different pore sizes (30–100 kDa) (Millipore, Billerica, MA). Although this method is very efficient for the recovery of double-stranded high-molecular-weight DNA free from PCR inhibitors, it is time-consuming, requires multiple tube transfers, and is difficult to automate.

Solid-Phase DNA Extraction Methods This extraction method is based on the ability of DNA to bind to silica in the presence of chaotropic salts such as guanidinium thiocyanate, sodium iodide, and guanidinium hydrochloride. Typically, cells are first lysed with proteinase K to release the DNA and then a binding buffer containing a chaotropic salt is added to prepare DNA for adsorption to the silica at pH < 7.5. Once DNA binds to silica, unwanted impurities can be rinsed away after subsequent washing steps, DNA may be eluted under alkaline conditions and low salt concentrations. The silica method can be carried out in two different formats: silica columns and silica-coated paramagnetic beads. In the first case, after DNA binding in the column, washing of impurities and DNA elution are made by centrifugation. In the magnetic beads procedure, washing steps and DNA elution are facilitated simply by applying a magnetic force without the need of centrifugation devices. Magnetic bead-based purification is currently one of the procedures best suited for DNA isolation in the forensic field as it enables rapid DNA purification with very efficient removal of PCR inhibitors, and it is suitable for high-throughput extraction using robotic platforms.

Chelating Resins (Chelex) A rapid and inexpensive procedure for DNA extraction that has become popular in the forensic field is the use of chelating resins, such as Chelex 100 (Bio-Rad Laboratories, CA). These resins can bind divalent ions such as Ca2þ and Mg2þ deactivating unwanted nucleases and, therefore, protecting DNA molecules from cleavage. In most protocols, forensic samples are added to a 5% Chelex suspension, boiled for several minutes, and then centrifuged to remove the resin leaving DNA in the supernatant. Unfortunately, the boiling procedure of chelating resins denatures DNA and yields single-stranded DNA that can be analyzed only by PCR-based methods. On the other hand, the DNA purity is not as good as that obtained with the organic extraction or the solid-phase procedures.

DNA from FTA Spots FTA® is an acronym for Fast Technology for Analysis of nucleic acids. It consists of a cellulose-based matrix treated with a weak

Encyclopedia of Forensic Sciences, Second Edition

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Biology/DNA | DNA Extraction and Quantification

Organic extraction Lysis (proteinase K, SDS)

Vortex with phenol chloroform

Solid-phase extraction

Lysis by boiling with 5% Chelex 100

Bind DNA to silica columns or beads

Centrifuge

Wash X 2 Transfer upper aqueous phase containing DNA to a new vial

Chelex extraction

Lysis (proteinase K, chaotropic salt)

Centrifuge

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Transfer the supernatant containing the DNA to a new vial

Elute DNA

Purify and concentrate DNA (filtration or ethanol precipitation)

Figure 1 Flowchart of most common Forensic DNA extraction methods.

base, a chelating agent, an anionic surfactant or detergent, and a uric acid (or a urate salt). Biological samples, such as blood or saliva, can be applied to FTA cards whose chemicals lyse cells and the released DNA remains immobilized. This system provides DNA preservation avoiding nuclease damage and microbial development, allowing a long-term storage at ambient temperature under dry conditions. FTA is at present a procedure widely implemented by several forensic laboratories for DNA collection of reference saliva or blood samples. There are two main strategies for DNA extraction from FTA paper. One is to wash out proteins and cellular debris from the FTA spot, keeping the DNA bound to the FTA, and then use a clean paper punch to perform the PCR analysis. Alternatively, DNA can be eluted from FTA by a Chelex extraction or other procedures using the eluted DNA for further analysis. The main advantages of FTA are the feasibility of automation and its long-term preservation due to its storage capabilities under ambient temperature.

Differential Lysis A specific protocol for the selective separation of epithelial cells DNA from sperm DNA in sexual assault cases was developed in 1985 by Peter Gill. The procedure is a modified version of the organic extraction method based on the resistance of sperm nuclei to be lysed in the absence of a reducing agent such as dithiothreitol (DTT). The protocol involves a first lysis step in the presence of SDS and proteinase K aimed to release the female epithelial cells DNA in the supernatant. The washed pellet of sperm cells are subsequently lysed by treatment with SDS, proteinase K, and DTT and the sperm DNA is recovered from the supernatant of this second lysis fraction. The success

of this method to separate sperm DNA from vaginal cell DNA depends on the relative number of each cell type and the conditions of preservation of the forensic evidence. Failure to separate the male and female fractions by this procedure results in a mixed DNA profile.

DNA Extraction from Bones and Teeth Several specific protocols have been described for DNA extraction from bones and teeth. All of them entail two primary steps. Previous preparation of compact bone tissue or teeth’s dentine powder by pulverization in liquid nitrogen using a freezer mill, and the use of high concentrations of ethylenediaminetetraacetic acid (EDTA) to demineralize the hydroxyapatite matrix making osteocytes or odontocytes accessible to lysis. Early forensic protocols performed demineralization of bone samples by extensive EDTA washes before the lysis step with the subsequent loss of cellular material during different washing steps and high risk of sample contamination. More recently, a number of protocols have been developed for complete demineralization during the lysis step (using a lysis buffer containing 0.5 M EDTA), resulting in full physical dissolution of the bone sample and maximal recovery of DNA. Bone or teeth powder lysates are then submitted to organic extraction followed by Amicon filtration or purified by silica solid-phase procedures.

Laser Capture Microdissection Laser capture microdissection (LCM) is a technique that allows to select and collect specific cell types. It is of particular interest

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Biology/DNA | DNA Extraction and Quantification

in the forensic field for specific sperm cell separation from mixtures of biological fluids in sexual assault cases. It combines existing light microscopic instrumentation with laser beam technology. There are two general methods of LCM: ultraviolet (UV) cutting systems and infrared (IR) capture systems. While in the IR system, after visualization of the cells of interest via microscopy, they are isolated by transfer of laser energy to a thermolabile polymer with formation of a polymer–cell composite. In the UV system, cells can be selectively captured by photovolatilization of cells surrounding the target cells. In both methods, captured sperm cells are transferred to a vial for DNA extraction. The LCM technique is used particularly in unbalanced mixtures in which very low levels of sperm cells are mixed with a high content of epithelial cells of the victim. As the number of captured cells is usually very small, the DNA extraction process is usually done by cell lysis in a small volume in the presence of proteinase K and a nonionic detergent such as Tween 20. Subsequent inactivation of the proteinase by a heat shock in the same vial of capture is carried out to finally obtain the DNA for downstream PCR analysis, minimizing the possibility of contamination and preventing the loss of DNA that could occur during the procedures for DNA purification.

Automation of DNA Extraction Development of robotic platforms for the extraction of DNA has been fundamental in ensuring a high-throughput processing of both reference samples and forensic evidences as well as guaranteeing reproducibility and sample tracking. Automation has been implemented in many forensic labs dealing with DNA profiling from large batches of reference samples for inclusion in national DNA databases. Automation has also taken great interest in disaster victim identification cases, enabling laboratories to speed up the process of DNA identification of missing persons. Most of the DNA extraction robots are based on solid-phase procedures with paramagnetic beads. There are several robotic stations for both small-scale and high-throughput processing as well as validated specific protocols for automated extraction of reference samples (blood, saliva, and FTA) and forensic samples (semen stains, blood, saliva, hair, bones, etc.). EZ1 (Qiagen), Maxwell 16 (Promega), and Automated Express (Life Science) are examples of small-scale platforms for automated DNA extraction of 6–16 samples simultaneously using paramagnetic beads, which have been validated for forensic samples. While the Tecan Freedom EVO automated liquidhandling workstation and the Beckman 2000 robot workstation are high-throughput platforms that can handle up to 96 samples at a time and are also validated for forensic analysis.

Microfluidic DNA Extraction Devices The development of miniaturized devices for DNA preparation, manipulation, and analysis at the micron (microtechnology) or submicron level (nanotechnology) has become one of the most active research areas in molecular biology. They offer several advantages over conventional techniques that include reduced sample and reagent consumption, high-throughput

and high-speed analysis, and easy automation and integration of different molecular analysis in a single biochip. In addition, microfabrication enables labs to increase the detection limit with the potential to manipulate DNA at the level of individual molecules with very important implications for the analysis of traditional challenges (DNA mixtures, low copy number, etc.) in forensic genetics. In respect to this technology, silica solidphase microchips have been developed for DNA extraction from forensic samples as well as some prototype microdevices for the differential lysis procedure.

DNA Quantification Quantification of human nuclear DNA from forensic samples is a recommended procedure (FBI Quality Assurance Standard 9.4) for a reliable DNA profiling based on multiplex PCR amplification and capillary electrophoresis detection of short tandem repeat (STR) markers. The first purpose of nuclear DNA quantification is to adjust the DNA input (around 0.5–1 ng of DNA template) in subsequent multiplex PCR– STRs assays for optimal performance. On the one hand, an adequate amount of DNA determination prevents PCR failures that are due to the absence of DNA or avoids STR–PCR artifacts, such as random allele dropout, produced by stochastic amplification effects from low-template DNA (LT-DNA) samples (under 100 pg of DNA). On the other hand, it prevents off-scale over amplification artifacts (including n  1 peaks, increased stutter bands and pullup) associated with an excess of DNA input in the PCR. In addition, an accurate DNA quantification helps to prevent the unnecessary waste of DNA, especially important when analyzing LT-DNA samples. Until recently, forensic laboratories have been using the slot-blot hybridization approach to target the D17Z1 locus, a highly repetitive alphoid primate-specific sequence, for DNA quantification in forensic casework. However, this methodology, with a detection limit above the limit of the STR profiling approaches, was often not sensitive enough to detect low-copy number forensic DNA samples. Moreover, the method is labor-intensive, time-consuming, and poorly suited to highthroughput sample flow. Several studies have demonstrated the usefulness of realtime PCR using Taqman probes or SYBR Green chemistry for sensitive, specific, and high-throughput DNA quantification assays using autosomal, X & Y chromosomes, and mitochondrial DNA targets. The development of commercially available real-time PCR human DNA quantification kits has also contributed to a worldwide use of real-time PCR in forensic genetics. Current DNA quantification kits are mainly based on three real-time PCR chemistries: the 50 nuclease activity assay using Taqman probes (Quantifiler Duo, Applied Biosystems), the Plexor chemistry using fluorescently labeled, iso-dC-containing primers (Plexor® HY System, Promega Corporation), or the use of Scorpion primers (Quantiplex Kit, Qiagen). Despite quantitation of total human DNA, these validated DNA assays offer qualitative data of great interest for forensic genetic typing: quantification of the presence of PCR inhibitors in the DNA extract, sex determination, and quantitative estimation of the proportion of the male component in mixtures of male and female biological fluids.

Biology/DNA | DNA Extraction and Quantification

Current Real-Time PCR Chemistries for Human DNA Quantification There are three commercial available fluorogenic chemistries to monitor the real-time progress of the PCR for the purpose of human DNA quantification that are validated for forensic samples: (1) by measuring the 50 nuclease activity of the Taq DNA polymerase to cleave a target-specific fluorogenic probe (a TaqMan probe: an oligonucleotide, complementary to a segment of the template DNA, with both a reporter and a quencher dye attached, that only emits its characteristic fluorescence after cleavage); (2) by measuring the decrease of fluorescence using one primer synthesized with an iso-dC residue as the 50 -terminal nucleotide linked to a fluorescent label and using dabcyl-iso-dGTP in the nucleotide mix (which pairs specifically with iso-dC) that quenches the signal of the fluorescent label primer when incorporates in the amplicons during the PCR; and (3) using scorpion primers that are bifunctional molecules containing a PCR primer covalently linked to a probe, which incorporates a fluorophore, and a quencher, which inhibits fluorescence. During PCR, when the probe binds to the PCR products, the fluorophore and quencher become separated leading to an increase in fluorescence. The application of double-stranded DNA-binding dye chemistry, such as SYBR Green, to DNA quantification from forensic samples has also been described. One drawback of SYBR Green-based detection is that nonspecific amplifications (primer–dimer, nonhuman products, etc.) cannot be distinguished from specific amplifications. On the other hand, the amplicon/dye ratio varies with amplicon length. Furthermore, SYBR green can only be used in singleplex PCRs. Real-time analysis of the fluorescence levels (increase or decrease depending on the chemistries) at each cycle of the PCR (amplification plot) allows obtaining a complete picture of the whole amplification process for each sample. In the initial cycles of PCR, a baseline is observed without any significant change in fluorescence signal. An increase in fluorescence above the baseline (or a decrease in fluorescence in case of Plexor HY System) indicates the detection of accumulated PCR product. The higher the initial input of the target genomic DNA, the sooner a significant increase (or decrease for Plexor HY System) in fluorescence is observed. The cycle at which fluorescence reaches an arbitrary threshold level during the exponential phase of the PCR is named Ct (threshold cycle). A standard curve can be generated by plotting the log of the starting DNA template amount of a set of previously quantified DNA standards against their Ct values. Therefore, an accurate estimation of the starting DNA amount from unknown samples is accomplished by comparison of the measured Ct values with the Ct values of the standard curve.

and one internal PCR control (IPC) (to evaluate PCR inhibition) using Taqman (Quantifiler Duo) or Plexor Chemistry (Promega). More recently, a new kit based on Scorpion primers chemistry (Qiagen) have been validated in the forensic field targeting an autosomal multicopy marker and also including one IPC. Table 1 shows the different targets included in these validated real-time PCR kits. Many other designs based on real-time PCR (mostly based on TaqMan chemistry) have been developed and validated in the forensic field to target single-copy autosomal markers, Alu repetitive elements, or X and Y chromosome-specific regions. The National Institute of Standards and Technology has developed a human DNA quantification standard (SRM 2372) intended for forensic applications that consists of three wellcharacterized human genomic DNA materials. The use of the same certified standard for human DNA quantification is expected to improve reproducibility across different laboratories and among different real-time PCR assays.

Real-Time PCR Mitochondrial DNA Quantification The use of a Taqman real-time PCR assay for quantification of mitochondrial human DNA (mtDNA) from forensic specimens was first described by Andre´asson et al. in 2002 by targeting a 142 bp region spanning over the genes for tRNA Lysine and ATP synthase 8 that can be amplified in a single PCR reaction or in combination with a nuclear DNA target. The specific quantification of human mtDNA by a Taqman realtime PCR assay of two different mtDNA fragment sizes (113 and 287 bp) within the hypervariable region I (HV1) of the mtDNA control region has also been described as a useful tool to evaluate the mtDNA preservation state (degradation) from ancient bone samples. A 69 bp fragment of the mtDNA NADH

Table 1 Targets included in real-time PCR forensic kits for nuclear human DNA quantification Real-time PCR kit

Human DNA target

Male DNA target

Internal PCR control

Quantifiler Duo

RPPH1 (ribonuclease PRNA component H1) 140 bp RNU2 locus (human U2 small nuclear RNA) 99 bp

SRY (sex-determining region Y) 130 bp

Artificial template 130 bp

TSPY gene (testis-specific protein Y encoded) 133 bp

Artificial template 150 bp

Plexor HY System

Quantiplex

Real-Time PCR Nuclear DNA Quantification Assays Two validated real-time PCR kits for human nuclear DNA quantification are currently used in the forensic DNA labs. Both kits allow simultaneous detection of one autosomal (single or multicopy) target (for total human DNA quantitation), a target of chromosome Y (for male human DNA quantitation),

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Proprietary region present on several autosomes of the human genome 146 bp

Artificial template 200 bp

Quantifiler Duo and Plexor HY System allow simultaneous quantitative analysis of human and male DNA, while Quantiplex only permits to detect a human multicopy DNA target. Three kits include an internal PCR control to monitor PCR inhibition. The size of each amplicon is shown in base pairs (bp).

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dehydrogenase subunit 1 (ND1) locus has also been used as a target for mtDNA quantitation in forensic specimens using a TaqMan duplex real-time PCR assay that allows simultaneous quantification of human nuclear DNA. Another mtDNA target used in forensics by a TaqMan-MGB assays is a 79-bp fragment of a conserved region of the human mtDNA, which is coamplified with an autosomal and a Y-chromosome target. Recently, it has been described a novel forensic utility of quantitative real-time PCR using TaqMan-MGB probes, targeting the highly variable mitochondrial single nucleotide polymorphism16519T/C to investigate heteroplasmic mixtures with an accurate quantification of the minor allele down to 9%. At this time, there is no commercially available real-time PCR assay for quantification of mtDNA nor has been developed a standard for mtDNA quantification in forensic materials. These seem to be some of the reasons why mtDNA quantitation is much less widespread in forensic casework.

Acknowledgments The author especially thanks Coro Ferna´ndez for proofreading of the manuscript and for her comments that have substantially improved the content of this article.

See also: Biology/DNA: Accreditation in Forensic DNA Analysis; Disaster Victim Identification; DNA Databases; Low-Template DNA Testing; Mixture Interpretation (Interpretation of Mixed DNA Profiles with STRs Only); Short Tandem Repeats.

Further Reading Alonso A and Garcı´a O (2007) Real-time quantitative PCR in forensic science. In: Rapley R and Whitehouse D (eds.) Molecular Forensics, pp. 59–71. New York: John Wiley & Sons Inc. Alonso A and Martı´n P (2005) A real-time PCR protocol to determine the number of amelogenin (X-Y) gene copies from forensic DNA samples. In: Carracedo A (ed.) Forensic DNA Typing Protocols Methods Mol Biol 297: 31–44. Alonso A, Martin P, Albarra´n C, et al. (2004) Real-time PCR designs to estimate nuclear and mitochondrial DNA copy number in forensic and ancient DNA studies. Forensic Science International 139: 141–149. Andreasson H, Gyllensten U, and Allen M (2002) Real-time DNA quantification of nuclear and mitochondrial DNA in forensic analysis. BioTechniques 33: 402–411. Anslinger K, Bayer B, Mack B, and Eisenmenger W (2007) Sex-specific fluorescent labelling of cells for laser microdissection and DNA profiling. International Journal of Legal Medicine 121: 54–56. Barbisin M, Fang R, O’Shea CE, Calandro LM, Furtado MR, and Shewale JG (2009) Developmental validation of the Quantifiler Duo DNA Quantification kit for simultaneous quantification of total human and human male DNA and detection of PCR inhibitors in biological samples. Journal of Forensic Sciences 54: 305–319.

Bienvenue JM, Duncalf N, Marchiarullo D, Ferrance JP, and Landers JP (2006) Microchip-based cell lysis and DNA extraction from sperm cells for application to forensic analysis. Journal of Forensic Sciences 51: 266–273. Bienvenue JM, Legendre LA, Ferrance JP, and Landers JP (2010) An integrated microfluidic device for DNA purification and PCR amplification of STR fragments. Forensic Science International Genetics 4: 178–186. Butler JM (2009a) DNA extraction. In: Butler JM (ed.) Fundamentals of Forensic DNA Typing, pp. 99–110. Amsterdam: Academic Press. Butler JM (2009b) DNA quantification. In: Butler JM (ed.) Fundamentals of Forensic DNA Typing, pp. 111–124. Amsterdam: Academic Press. Gill P, et al. (1985) Forensic applications of DNA fingerprints. Nature 318: 577–579. Hochmeister MN, Budowle B, Borer UV, Eggmann U, Comey CT, and Dirnhofer R (1991) Typing of deoxyribonucleic acid (DNA) extracted from compact bone from human remains. Journal of Forensic Sciences 36: 1649–1661. Horsman KM, Bienvenue JM, Blasier KR, and Landers JP (2007) Forensic DNA analysis on microfluidic devices: A review. Journal of Forensic Sciences 52: 784–799. Kline MC, Duewer DL, Travis JC, et al. (2009) Production and certification of NIST Standard Reference Material 2372 Human DNA Quantitation Standard. Analytical and Bioanalytical Chemistry 394: 1183–1192. Loreille OM, Diegoli TM, Irwin JA, Coble MD, and Parsons TJ (2007) High efficiency DNA extraction from bone by total demineralization. Forensic Science International Genetics 1: 191–195. Murray C, McAlister C, and Elliott K (2007) Identification and isolation of male cells using fluorescence in situ hybridisation and laser microdissection, for use in the investigation of sexual assault. Forensic Science International Genetics 1: 247–252. Nagy M (2007) Automated DNA extraction techniques for forensic analysis. In: Rapley R and Whitehouse D (eds.) Molecular Forensics, pp. 37–58. New York: John Wiley & Sons Inc. Nicklas JA and Buel E (2003) Development of an Alu-based, real-time PCR method for quantitation of human DNA in forensic samples. Journal of Forensic Sciences 48: 936–944. Swango KL, Hudlow WR, Timken MD, and Buoncristiani MR (2007) Developmental validation of a multiplex qPCR assay for assessing the quantity and quality of nuclear DNA in forensic samples. Forensic Science International 170: 35–45. Timken MD, Swango KL, Orrego C, and Buoncristiani MR (2005) A duplex real-time qPCR assay for the quantification of human nuclear and mitochondrial DNA in forensic samples: Implications for quantifying DNA in degraded samples. Journal of Forensic Sciences 50: 1044–1060. Vandewoestyne M and Deforce D (2010) Laser capture microdissection in forensic research: A review. International Journal of Legal Medicine 124: 513–521. Walker JA, Hedges DJ, Perodeau BP, et al. (2005) Multiplex polymerase chain reaction for simultaneous quantitation of human nuclear, mitochondrial, and male Y-chromosome DNA: Application in human identification. Analytical Biochemistry 337: 89–97.

Relevant Websites http://marketing.appliedbiosystems.com – Applied Biosystems Prepfiler system. http://www.dna.gov/training – DNA Initiative. DNA Extraction and Quantification. http://www.dna.gov – DNA Initiative. Human DNA Quantitation. http://www.cstl.nist.gov – DNA Quantitation Efforts by the NIST Forensics/Human Identity Project Team. https://www.promega.com – Plexor HY System. http://www.promega.com – Promega DNA IQ system. http://www.qiagen.com – Qiagen DNA Extraction methods in forensic. http://marketing.appliedbiosystems.com – QuantifilerDuo. http://www.qiagen.com – Quantiplex.