Full Short Tandem Repeat Profiles Research Articles

Assessment of body fluid identification and DNA profiling after exposure to tropical weather conditions.

Despite current advances in body fluid identification, there are few studies evaluating the effect of environmental conditions. The present work assessed the detection of body fluids, blood, semen, and saliva, through lateral flow immunochromatographic (LFI) tests, exposed to tropical weather conditions over time, also evaluating the possibility of obtaining STR (short tandem repeat) profiles and identifying mitochondrial DNA (mtDNA) polymorphisms. Blood, semen, saliva samples, and mixtures of these fluids were deposited on polyester clothes and exposed to open-air tropical weather conditions for 1 month. The test versions from LFI (SERATEC®, Germany) Lab and crime scene (CS) used for the detection - one per each body fluid type - demonstrated that it is possible to identify body fluids and their mixtures up to 14 days after deposition. At 30 days, blood and semen were detected but not saliva. Full STR profiles were obtained from 14-day-old blood samples, and partial profiles were obtained from the remaining samples. It was possible to sequence mtDNA in the samples previously analyzed for STR profiling, and haplogroups could be assigned. In conclusion, this study demonstrated for the first time the possibility of body fluid identification and DNA profiling after exposure to tropical weather conditions for 1 month and also demonstrated the value of mtDNA analysis for compromised biological evidence.

Degradation of nuclear and mitochondrial DNA after γ-irradiation and its effect on forensic genotyping.

Forensic genotyping can be impeded by γ-irradiation of biological evidence in the event of radiological crime; that is, criminal activity involving radioactive material. Oxidative effects within the mitochondria of living cells elicits greater damage to mitochondrial DNA (mtDNA) than nuclear DNA (nuDNA) at low doses. This study presents a novel approach for the assessment of nuDNA versus mtDNA damage from a comparison of genotype and quantity data, while exploring likely mechanisms for differential damage after high doses of γ-irradiation. Liquid (hydrated) and dried (dehydrated) whole blood samples were exposed to high doses of γ-radiation (1-50 kilogray, kGy). The GlobalFiler PCR Amplification Kit was used to evaluate short tandem repeat (STR) genotyping efficacy and nuDNA degradation; a comparison was made to mtDNA degradation measured using real-time PCR assays. Each assay was normalized before comparison by calculation of integrity indices relative to unirradiated controls. Full STR profiles were attainable up to the highest dose, although DNA degradation was noticeable after 10 and 25kGy for hydrated and dehydrated blood, respectively. This was manifested by heterozygote imbalance more than allele dropout. Degradation was greater for mtDNA than nuDNA, as well as for hydrated than dehydrated cells, after equivalent doses. Oxidative effects due to water radiolysis and mitochondrial function are dominant mechanisms of differential damage to nuDNA versus mtDNA after high-dose γ-irradiation. While differential DNA damage was reduced by cell desiccation, its persistence after drying indicates innate differences between nuDNA and mtDNA radioresistance and/or continued oxidative effects within the mitochondria.

A quantitative method for selecting a hair for nuclear DNA analysis

This study evaluated a quantitative method to predict the success of nuclear DNA (nuDNA) typing for head hair roots, using the minor-groove DNA binding dye, 4′, 6-diamidino-2-phenylindole (DAPI). The procedure was successful in staining nuclear material in hair roots, regardless of soft tissue presence or growth phase. We found that the dye can even reveal an abundance of visible nuclei in hairs that were previously assumed to be unsuitable for nuDNA analysis (e.g., telogen hairs). The value of DAPI staining is particularly evident when considering the STR typing results for telogen hairs. Here, telogen hairs with greater than 100 visible nuclei frequently produced full or high-partial STR profiles, while telogen hairs with fewer than 100 visible nuclei rarely resulted in >20 % STR allele recovery. In addition, our findings indicated no interference by DAPI in the forensic examination of hair evidence, including preparation of hairs on microscopic slides, microscopic examination, DNA extraction, quantitative PCR, and short tandem repeat (STR) typing. Furthermore, the method remained steadfast for hairs washed by sonication as well as hairs retrieved from Permount™ mounting medium. When validated, this simple, quick, and quantitative screening method can be used in casework to select a hair for nuDNA analysis, especially for hairs that were previously sent directly for mitochondrial (mt) DNA analysis based on the lack of adhering soft tissue, regardless of growth phase. Conversely, nuDNA degradation may exist in hairs which exhibit microscopic characteristics typically associated with a potential to generate successful nuclear DNA profile including stretched roots with attached root sheath. DAPI staining of hairs gives forensic examiners the ability to have more information, other than growth phase, when selecting a hair or hairs for possible nuDNA analysis.

Forensic identification of missing persons using DNA from surviving relatives and femur bone retrieved from salty environment

Human identification using forensic DNA profiling has made enormous advancement over the past two-and-half decades. Forensic DNA profiling provides enormous genetic data from a variety of biological materials and individuals to help solve many important criminal and civil cases that confront society. Under certain environmental conditions, the total deterioration of soft-tissue leaves skeletal remains as the only available sample for DNA testing to identify missing persons, victims of natural disasters, or exonerate suspect(s) in a criminal case. We report the findings of a case involving the human remains of a missing person submitted to the Forensic Science Laboratory of the Ghana Police Service for forensic DNA profiling in comparison to an alleged living relative of the deceased. DNA from the femur bone and buccal swabs of alleged relative of the deceased were extracted, quantified, and short tandem repeat (STR) profiled using Qiagen's Investigator kit, Applied Biosystem's Quantifiler trio, and GlobalFiler kits. Full STR profiles were generated for both the femur bone from the salty environment and the buccal swabs from the alleged relative. The femur bone was genetically identified to be that of the missing person. The remains were thus handed over to the relatives for final funeral rites and burial to bring closure to the long search for the missing person.

Modular-Based Integrated Microsystem with Multiple Sample Preparation Modules for Automated Forensic DNA Typing from Reference to Challenging Samples.

The realization of an automated short tandem repeat (STR) analysis for forensic investigations is facing a unique challenge, that is DNA evidence with wide disparities in sample types, quality, and quantity. We developed a fully integrated microsystem in a modular-based architecture to accept and process various forensic samples in a "sample-in-answer-out" manner for forensic STR analysis. Two sample preparation modules (SPMs), the direct and the extraction SPM, were designed to be easily assembled with a capillary array electrophoresis (CAE) chip using a chip cartridge to efficiently achieve an adequate performance to different samples at a low cost. The direct SPM processed buccal swabs to produce STR profiles without DNA extraction in about 2 h. The extraction SPM analyzed more challenging blood samples based on chitosan-modified quartz filter paper for DNA extraction. This newly developed quartz filter provided a 90% DNA extraction efficiency and the "in situ" PCR capability, which enabled DNA extraction and PCR performed within a single chamber with all the DNA concentrated in the filter. We demonstrated that minute amounts of blood (0.25 μL), highly diluted blood (0.5 μL blood in 1 mL buffer), and latent bloodstains (5-μL bloodstain on cloth washed with detergent) can be automatically analyzed using our microsystem, reliably producing full STR profiles with a 100% calling of all the alleles. This modular-based microsystem with the capability of analyzing a wide range of samples should be able to play an increasing role in both urgent situations and routine forensic investigations, dramatically extending the applications and utility of automated DNA typing.

Increased recovery of touch DNA evidence using FTA paper compared to conventional collection methods

Tape lifting and FTA paper scraping methods were directly compared to traditional double swabbing for collecting touch DNA from car steering wheels (n = 70 cars). Touch DNA was collected from the left or right side of each steering wheel (randomized) using two sterile cotton swabs, while the other side was sampled using water-soluble tape or FTA paper cards. DNA was extracted and quantified in duplicate using qPCR. Quantifiable amounts of DNA were detected for 100% of the samples (n = 140) collected independent of the method. However, the DNA collection yield was dependent on the collection method. A statistically significant difference in DNA yield was observed between FTA scraping and double swabbing methods (p = 0.0051), with FTA paper collecting a two-fold higher amount. Statistical analysis showed no significant difference in DNA yields between the double swabbing and tape lifting techniques (p = 0.21).Based on the DNA concentration required for 1 ng input, 47% of the samples collected using FTA paper would be expected to yield a short tandem repeat (STR) profile compared to 30% and 23% using double swabbing or tape, respectively. Further, 55% and 77% of the samples collected using double swabbing or tape, respectively, did not yield a high enough DNA concentration for the 0.5 ng of DNA input recommended for conventional STR kits and would be expected to result in a partial or no profile compared to 35% of the samples collected using FTA paper. STR analysis was conducted for a subset of the higher concentrated samples to confirm that the DNA collected from the steering wheel was from the driver. 32 samples were selected with DNA amounts of at least 1 ng total DNA (100 pg/μl when concentrated if required). A mixed STR profile was observed for 26 samples (88%) and the last driver was the major DNA contributor for 29 samples (94%). For one sample, the last driver was the minor DNA contributor. A full STR profile of the last driver was observed for 21 samples (69%) and a partial profile was observed for nine samples (25%); STR analysis failed for two samples collected using tape (6%).In conclusion, we show that the FTA paper scraping method has the potential to collect higher DNA yields from touch DNA evidence deposited on non-porous surfaces often encountered in criminal cases compared to conventional methods.

A fully integrated microchip system for automated forensic short tandem repeat analysis.

We have successfully developed an integrated microsystem that combines two plastic microchips for DNA extraction and PCR amplification with a glass capillary array electrophoresis chip together in a compact control and detection instrument for automated forensic short tandem repeat (STR) analysis. DNA extraction followed by an "in situ PCR" was conducted in a single reaction chamber of the microchip based on a filter paper-based extraction methodology. PCR products were then mixed with sizing standards by an injection electrode and injected into the electrophoresis chip for four-color confocal fluorescence detection. The entire STR analysis can be completed in about two hours without any human intervention. Since the 15-plex STR system has a more stringent requirement for PCR efficiency, we optimized the structure of the plastic DNA extraction and amplification chip, in which the reaction chamber was formed by sandwiching a hollow structure layer with two blank cover layers, to reduce the adsorption of PCR reagents to the surfaces. In addition, PCR additives, bovine serum albumin, poly(ethylene glycol), and more magnesium chloride were included into the on-chip multiplex STR system. The limit-of-detection study demonstrated that our microsystem was able to produce full 15-plex STR profiles from 3.75 ng standard K562 DNA. Buccal swab and whole blood samples were also successfully typed by our system, validating the feasibility of performing rapid DNA typing in a "sample-in-answer-out" manner for on-site forensic human identification.

Preservation of and DNA Extraction from Muscle Tissue.

As well as protecting DNA for subsequent analysis, tissue preservation methods ideally should be safe, readily available, and easy to transport at relatively low cost. Formalin (formaldehyde solution), used extensively to preserve medical and museum specimens, irreparably damages DNA. We have found four tissue preservatives (solid salt, salt-saturated dimethyl sulfoxide (DMSO)-EDTA solution, ethanol solution, and ethanol-EDTA solution) that preserved muscle tissue at 35 °C for up to 1 month: full short tandem repeat (STR) profiles were obtained after preservation. In addition, salt-saturated DMSO-EDTA solution yielded full STR profiles from aliquots of the liquid preservative surrounding muscle tissue.

Identification of Colored Dyes that are Resistant to Fading on Exposure to Ethylene Oxide; Use with Indicating FTA™ Sample Collection Cards

Regulatory Standards and Forensic Communities are expressing an expectation for HID products to be certified as “DNA-free.” Recently, “DNA-free” status was described for HID-related products using ethylene oxide (EtO); this gas reduces the presence of amplifiable DNA and causes minimal interference to downstream HID-analytical methods. During sample collection, indicating cards, for example, Indicating FTA™ (GE Healthcare Life Sciences, UK), are used to collect and store buccal cell DNA. These cards contain a dye which changes color on application of a colorless sample. Generating “DNA-free” indicating cards using EtO should not impact the dyes' ability to indicate sample location or the efficacy of the card in downstream HID-analytical methods. This study was initiated to identify alternative dyes to those currently used with sample indicating collection cards. The most promising, dyes when applied to cellulose papers exhibited a uniform color distribution and excellent sample indicating properties even when mixed with chemicals associated with FTA™. When dyed cellulose papers were exposed to EtO, ultraviolet radiation, elevated temperature, and humidity, negligible fading or discoloration was observed. The presence of these dyes on cellulose papers did not interfere with direct short tandem repeat (STR) profiling. Allelic concordance, first pass success rate, and mean peak heights were comparable to samples applied to Indicating FTA. Biological samples applied to EtO-treated dyed cellulose papers and stored >1 month produced full STR profiles of sufficient quality to allow submission to DNA databases, confirming negligible interference from EtO treatment. These alternative sample indicating dyes resist EtO-mediated fading while fulfilling the Forensic Community's expectation for “DNA-free” with negligible impact on collection card performance.

Temporal analysis of 16 STR loci in human blood drawn from two culicid mosquitoes cultured at different temperatures

AbstractFemale mosquitoes feed on human blood, which can be collected to analyze human short tandem repeat (STR) sequences; these are specific to each human individual. Analysis of STRs might help in identification of a person found near a crime scene. Aedes aegypti and Culex pipiens mosquitoes fed on human blood were cultured at 18°C or 40°C (median temperature for summer and winter time in Riyadh governorate, Saudi Arabia) for 3, 6, 12, 24, 48 and 72 h. In A. aegypti, human DNA concentration was reduced with time at both temperatures. At 18°C, we obtained full STR profiles up to 48 h post feeding on human blood while none of the 16 loci were obtained at 72 h. At 40°C, we missed six sites at 12 h after blood sucking, 12 at 24 h, and 15 at 48 h and 72 h. In C. pipiens cultured at 18°C, full profiles were developed up to 48 h following blood feeding while we could not amplify five sites at 72 h. At 40°C, mortality among females was 50% at 24 h and 100% at both 48 h and 72 h; however, we had full profiles in all samples including dead insects. This research addressed the possibility of using mosquitoes in forensic research by DNA genotyping by changing the mosquito culturing temperature and mosquito genus. Our findings proved that different types of mosquito change the temporal pattern of STR analysis and showed that the mosquito culturing temperature affects the integrity of DNA for STR analysis.

The Potential of Cosmetic Applicators as a Source of DNA for Forensic Analysis.

Personal products, such as toothbrushes, have been used as both known reference and evidentiary samples for forensic DNA analysis. This study examined the viability of a broad selection of cosmetic applicators for use as targets for human DNA extraction and short tandem repeat (STR) analysis using standard polymerase chain reaction (PCR) conditions. Applicator types included eyeliner smudgers, pencils and crayons, eye shadow sponges, mascara wands, concealer wands, face makeup sponges, pads and brushes, lipsticks and balms, and lip gloss wands. The quantity and quality of DNA extracted from each type of applicator were examined by assessing the number of loci successfully amplified and the peak balance of the heterozygous alleles in each full STR profile. While degraded DNA, stochastic amplification, and PCR inhibition were observed for some items, full STR profiles were developed for 14 of 76 applicators. The face makeup sponge applicators yielded the highest proportional number of full STR profiles (4/7).

The 'triple contrast' method in experimental wound ballistics and backspatter analysis.

In practical forensic casework, backspatter recovered from shooters' hands can be an indicator of self-inflicted gunshot wounds to the head. In such cases, backspatter retrieved from inside the barrel indicates that the weapon found at the death scene was involved in causing the injury to the head. However, systematic research on the aspects conditioning presence, amount and specific patterns of backspatter is lacking so far. Herein, a new concept of backspatter investigation is presented, comprising staining technique, weapon and target medium: the 'triple contrast method' was developed, tested and is introduced for experimental backspatter analysis. First, mixtures of various proportions of acrylic paint for optical detection, barium sulphate for radiocontrast imaging in computed tomography and fresh human blood for PCR-based DNA profiling were generated (triple mixture) and tested for DNA quantification and short tandem repeat (STR) typing success. All tested mixtures yielded sufficient DNA that produced full STR profiles suitable for forensic identification. Then, for backspatter analysis, sealed foil bags containing the triple mixture were attached to plastic bottles filled with 10% ballistic gelatine and covered by a 2-3-mm layer of silicone. To simulate backspatter, close contact shots were fired at these models. Endoscopy of the barrel inside revealed coloured backspatter containing typable DNA and radiographic imaging showed a contrasted bullet path in the gelatine. Cross sections of the gelatine core exhibited cracks and fissures stained by the acrylic paint facilitating wound ballistic analysis.

Efficiency of Buccal DNA Sampling Device in the Mortuary

Identification of forensic DNA samples by short tandem repeat (STR) profiling is currently an essential component of criminal investigations and can aid in linking perpetrators to crimes as well as identifying missing individuals or unidentified remains. In South Africa, recent amendments to legislation have allowed for the mandatory acquisition of reference DNA samples from certain offenders in order to populate the new National Forensic DNA Database. A novel method for the collection of buccal samples, the EasiCollect device, has been proposed to facilitate the collection of these DNA samples, replacing blood collecting devices as the standard method of DNA collection. Subsequently, this device has been introduced into South African state mortuaries to assist in the identification of deceased individuals. In order to ascertain if this device is suitable for use in the postmortem setting, an investigation was performed to compare the main methodology currently utilised within South African mortuaries, namely femoral blood transferred to ‘Fast Technology for Analysis of nucleic acids’ (FTA) cards, and buccal cells obtained using the EasiCollect device. DNA yields and STR genotyping results were compared between the two collection methods in thirty deceased individuals. Buccal samples provided a significantly greater DNA yield than blood samples, while no significant difference was observed between the qualities of the sample types as measured by the 260/280 nm ratio. Full STR profiles were obtained from all blood and buccal samples, with amplification efficiency showing limited DNA degradation and PCR inhibition in these samples, and only 3% of samples giving potentially disputable results. Numerous issues surrounding the collection of blood samples, however, indicated that this method is not optimal for use in the mortuary, with the EasiCollect device providing a more practical and robust method for the collection of DNA samples in the mortuary.

Minimizing inhibition of PCR-STR typing using digital agarose droplet microfluidics.

The presence of PCR inhibitors in forensic and other biological samples reduces the amplification efficiency, sometimes resulting in complete PCR failure. Here we demonstrate a high-performance digital agarose droplet microfluidics technique for single-cell and single-molecule forensic short tandem repeat (STR) typing of samples contaminated with high concentrations of PCR inhibitors. In our multifaceted strategy, the mitigation of inhibitory effects is achieved by the efficient removal of inhibitors from the porous agarose microgel droplets carrying the DNA template through washing and by the significant dilution of targets and remaining inhibitors to the stochastic limit within the ultralow nL volume droplet reactors. Compared to conventional tube-based bulk PCR, our technique shows enhanced (20 ×, 10 ×, and 16 ×) tolerance of urea, tannic acid, and humic acid, respectively, in STR typing of GM09948 human lymphoid cells. STR profiling of single cells is not affected by small soluble molecules like urea and tannic acid because of their effective elimination from the agarose droplets; however, higher molecular weight humic acid still partially inhibits single-cell PCR when the concentration is higher than 200 ng/μL. Nevertheless, the full STR profile of 9948 male genomic DNA contaminated with 500 ng/μL humic acid was generated by pooling and amplifying beads carrying single-molecule 9948 DNA PCR products in a single secondary reaction. This superior performance suggests that our digital agarose droplet microfluidics technology is a promising approach for analyzing low-abundance DNA targets in the presence of inhibitors.

Ultrafast Amplification of DNA on Plastic Microdevices for Forensic Short Tandem Repeat Analysis

The majority of microfluidic devices used as a platform for low-cost, rapid DNA analysis are glass devices; however, microchip fabrication in glass is costly and laborious, enhancing the interest in polymeric substrates, such as poly (methyl methacrylate) (PMMA), as an inexpensive alternative. Here, we report amplification in PMMA polymerase chain reaction (PCR) microchips providing full short tandem repeat profiles (16 of 16 loci) in 30-40 min, with peak height ratios and stutter percentages that meet literature threshold requirements. In addition, partial profiles (15 of 16 loci) were generated using an ultrafast PCR method in 17.1 min, representing a ~10-fold reduction in reaction time as compared to current amplification methods. Finally, a multichamber device was demonstrated to simultaneously amplify one positive, one negative, and five individual samples in 39 min. Although there were instances of loci dropout, this device represents a first step toward a microfluidic system capable of amplifying more than one sample simultaneously.

An enzyme-based DNA preparation method for application to forensic biological samples and degraded stains

Extraction of DNA from forensic samples typically uses either an organic extraction protocol or solid phase extraction (SPE) and these methods generally involve numerous sample transfer, wash and centrifugation steps. Although SPE has been successfully adapted to the microdevice, it can be problematic because of lengthy load times and uneven packing of the solid phase. A closed-tube enzyme-based DNA preparation method has recently been developed which uses a neutral proteinase to lyse cells and degrade proteins and nucleases [14]. Following a 20min incubation of the buccal or whole blood sample with this proteinase, DNA is polymerase chain reaction (PCR)-ready. This paper describes the optimization and quantitation of DNA yield using this method, and application to forensic biological samples, including UV- and heat-degraded whole blood samples on cotton or blue denim substrates. Results demonstrate that DNA yield can be increased from 1.42 (±0.21)ng/μL to 7.78 (±1.40)ng/μL by increasing the quantity of enzyme per reaction by 3-fold. Additionally, there is a linear relationship between the amount of starting cellular material added and the concentration of DNA in the solution, thereby allowing DNA yield estimations to be made. In addition, short tandem repeat (STR) profile results obtained using DNA prepared with the enzyme method were comparable to those obtained with a conventional SPE method, resulting in full STR profiles (16 of 16 loci) from liquid samples (buccal swab eluate and whole blood), dried buccal swabs and bloodstains and partial profiles from UV or heat-degraded bloodstains on cotton or blue denim substrates. Finally, the DNA preparation method is shown to be adaptable to glass or poly(methyl methacrylate) (PMMA) microdevices with little impact on STR peak height but providing a 20-fold reduction in incubation time (as little as 60s), leading to a ≥1h reduction in DNA preparation time.

Persistence of biological traces in gun barrels—an approach to an experimental model

Traces of backspatter in gun barrels after homicidal or suicidal contact shots may be a valuable source of forensic evidence. Yet, a systematic investigation of the persistence and durability of DNA from biological traces in gun barrels is lacking. Our aim was to generate a realistic model to emulate blood and tissue spatters in gun barrels generated by contact gunshots at biological targets and to analyse the persistence and typability of DNA recovered from such stains. Herein, we devise and evaluate three different models for the emulation of backspatter from contact shots: a gelatine-based model with embedded blood bags, a model based on a spongious matrix soaked with blood and covered with a thin plastic membrane and a head model consisting of an acrylic half sphere filled with ballistic gelatine and with blood bags attached to the sphere under a 3-mm silicone layer. The sampling procedure for all three models: a first shot was fired with several types of guns at each model construction and subsequently a second shot was fired at a backstop. Blood samples were collected after each shot by probing the inner surface of the front and rear end of the respective gun barrel with a sterile swab. DNA was then extracted and quantified and up to 20 different short tandem repeat (STR) systems were amplified to generate DNA profiles. Although DNA quantity and STR typing results were heterogenous between the models, all models succeeded in delivering full STR profiles even after more than one shot. We conclude that biological traces in gun barrels are robust and accessible to forensic analysis and that systematic examination of the inside of gun barrels may be advisable for forensic casework.

The Use of Hemastix® Severely Reduces DNA Recovery Using the BioRobot® EZ1

The choice of reagents for presumptive tests for blood, and subsequent extraction methodologies, can significantly affect both the quantity and quality of purified DNA. Blood samples directly tested with Hemastix(®) yielded <1% of the DNA recovered from untested samples when purified using the Qiagen BioRobot(®) EZ1 and EZ1(®) DNA Investigator kit. Full short tandem repeat profiles were obtained from both tested and untested samples, suggesting that the Hemastix(®) reagent(s) affect DNA binding, rather than produce DNA damage. The Hemastix(®) inhibition of DNA yield could be overcome by the addition of MTL buffer to the sample prior to extraction. Laboratories may wish to modify current procedures for extracting blood samples, utilize other extraction/purification methodologies, or inform their submitting agencies to avoid direct exposure of questioned bloodstains to Hemastix(®) reagents.

A valveless microfluidic device for integrated solid phase extraction and polymerase chain reaction for short tandem repeat (STR) analysis

A valveless microdevice has been developed for the integration of solid phase extraction (SPE) and polymerase chain reaction (PCR) on a single chip for the short tandem repeat (STR) analysis of DNA from a biological sample. The device consists of two domains--a SPE domain filled with silica beads as a solid phase and a PCR domain with an ~500 nL reaction chamber. DNA from buccal swabs was purified and amplified using the integrated device and a full STR profile (16 loci) resulted. The 16 loci Identifiler® multiplex amplification was performed using a non-contact infrared (IR)-mediated PCR system built in-house, after syringe-driven SPE, providing an ~80-fold and 2.2-fold reduction in sample and reagent volumes consumed, respectively, as well as an ~5-fold reduction in the overall analysis time in comparison to conventional analysis. Results indicate that the SPE-PCR system can be used for many applications requiring genetic analysis, and the future addition of microchip electrophoresis (ME) to the system would allow for the complete processing of biological samples for forensic STR analysis on a single microdevice.

Integrated DNA purification, PCR, sample cleanup, and capillary electrophoresis microchip for forensic human identification

A fully integrated microdevice and process for forensic short tandem repeat (STR) analysis has been developed that includes sequence-specific DNA template purification, polymerase chain reaction (PCR), post-PCR cleanup and inline injection, and capillary electrophoresis (CE). Fragmented genomic DNA is hybridized with biotin-labeled capture oligos and pumped through a fluidized bed of magnetically immobilized streptavidin-coated beads in microchannels where the target DNA is bound to the beads. The bead-DNA conjugates are then transferred into a 250 nL PCR reactor for autosomal STR amplification using one biotin and one fluorescence-labeled primer. The resulting biotin-labeled PCR products are electrophoretically injected through a streptavidin-modified capture gel where they are captured to form a concentrated and purified injection plug. The thermally released sample plug is injected into a 14 cm long CE column for fragment separation and detection. The DNA template capture efficiency provided by the on-chip sequence-specific template purification is determined to be 5.4% using K562 standard DNA. This system can produce full 9-plex STR profiles from 2.5 ng input standard DNA and obtain STR profiles from oral swabs in about 3 hours. This fully integrated microsystem with sample-in-answer-out capability is a significant advance in the development of rapid, sensitive, and reliable micro-total analysis systems for on-site human identification.