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.     

Automated magnetic preparation of DNA templates for solid phase sequencing.

An integrated protocol for solid-phase DNA sequencing using a robotic work station is described involving magnetic separation of DNA and analysis of the sequencing product by electrophoresis with automated detection of the fluorescently labeled fragments. The method, which is based on magnetic beads in combination with streptavidin-biotin technology, can be used for sequencing both genomic and plasmid DNA. The DNA template is obtained by the polymerase chain reaction (PCR). Protocols to prepare five and ten immobilized samples is described, giving 10 and 20 single-stranded templates, respectively. The magnetic purification steps are performed in a microtiter plate and this allows for an integrated scheme involving a subsequent procedure for automated primer annealing and sequencing reactions. Here, the procedure is examplified by direct genomic sequencing of DNA in blood sample from a human immunodeficiency virus (HIV)-infected patient and a cloned human antibody DNA fragment using fluorescently labeled sequencing primers.     

Development of an automated DNA purification module using a micro-fabricated pillar chip

We present a fully automated DNA purification module comprised of a micro-fabricated chip and sequential injection analysis system that is designed for use within autonomous instruments that continuously monitor the environment for the presence of biological threat agents. The chip has an elliptical flow channel containing a bed (3.5 x 3.5 mm) of silica-coated pillars with height, width and center-to-center spacing of 200, 15, and 30 microm, respectively, which provides a relatively large surface area (ca. 3 cm(2)) for DNA capture in the presence of chaotropic agents. We have characterized the effect of various fluidic parameters on extraction performance, including sample input volume, capture flow rate, and elution volume. The flow-through design made the pillar chip completely reusable; carryover was eliminated by flushing lines with sodium hypochlorite and deionized water between assays. A mass balance was conducted to determine the fate of input DNA not recovered in the eluent. The device was capable of purifying and recovering Bacillus anthracis genomic DNA (input masses from 0.32 to 320 pg) from spiked environmental aerosol samples, for subsequent analysis using polymerase chain reaction-based assays.     

Detection of genetically modified organisms in food: critical points for quality assurance

 The detection of genetically modified organisms (GMOs) by the polymerase chain reaction (PCR) is a complex multiparameter problem. Therefore, a number of critical issues in respect to quality control need to be considered. For practical purposes, the PCR process itself can be divided into three subprocesses: template isolation and reaction setup (pre-PCR), PCR reaction and detection of amplification products, and data evaluation (post-PCR). Crucial factors for the pre-PCR process are the following: homogeneity of the sample to be analysed, performance of template isolation and purification in terms of yield and purity, standardized process for the estimation of concentrations of genomic DNA and all reagents used in the reaction. For the PCR itself, crucial factors to be controlled are: setup of reactions, batch to batch variations of reagents, temperature-time programs used for the PCR amplification, and the performance of different types of hardware (e.g. different brands of thermocyclers). The crucial factor for the post-PCR process is the detection of the amplification products of the PCR. The tremendous sensitivity of PCR methods requires a careful and consequent separation of the three processes in terms of hardware, laboratory space and sample handling. The avoidance of contamination is one of the most critical factors. The goal of quality assurance measures must be to ensure appropriate results at maximum sensitivity. The complexity of any PCR system used for the detection of GMOs leads to the requirement of a careful validation process for any laboratory using such methods. For qualitative analyses crucial validation parameters are: specificity, selectivity, repeatability, intermediate precision, reproducibility, limit of detection and robustness. Received: 5 October 1998 / Accepted: 22 February 1999     

Developments toward a complete micro-total analysis system for Duchenne muscular dystrophy diagnosis

The diagnosis of Duchenne muscular dystrophy (DMD) has historically utilized either PCR or requires Southern blot analysis, a southern blot analysis, however, is not amenable to incorporation in a microdevice format. A PCR amplification-based method has been developed, and we have previously coupled this amplification with microchip separation of the PCR fragments for DMD diagnosis. Diagnoses of affected patients were performed by comparing exon concentrations to those of control samples amplified at the same time. To accurately identify mutations in patient samples, this work established normal ranges for the concentration of each amplified exon fragment using control samples amplified over successive days. Our studies show that the number of cycles used in the amplification process affects this range. Affected patient samples were analyzed using these normal ranges and the mutations detected by Southern blot analysis were also diagnosed using the microchip separation method.

Employing the microchip separation method decreases the time required for the analysis, but the time required for DNA purification and PCR amplification must also be decreased for faster total analysis of patient samples. Development of microchip methods for these processing steps is one approach for reducing the individual times, while also providing the possibility of integrating these steps in a single device. Here we report on the microchip extraction of genomic DNA from whole blood using a novel sol–gel matrix that is easily formed in microdevices. IR-mediated PCR amplification of a β-globin fragment from genomic DNA followed by electrophoretic analysis on a single integrated microdevice is presented for the first time. Work towards the development of a micro-total analysis device for DMD diagnosis, through integration of all processing steps on a single device, is also discussed.     

One-Step Nucleic Acid Purification and Noise-Resistant Polymerase Chain Reaction by Electrokinetic Concentration for Ultralow-Abundance Nucleic Acid Detection

Nucleic acid amplification tests (NAATs)integrated on a chip hold great promise for point-of-care diagnostics. Currently, nucleic acid (NA) purification remains time-consuming and labor-intensive, and it takes extensive efforts to optimize the amplification chemistry. Using selective electrokinetic concentration, we report one-step, liquid-phase NA purification that is simpler and faster than conventional solid-phase extraction. By further re-concentrating NAs and performing polymerase chain reaction (PCR) in a microfluidic chamber, our platform suppresses non-specific amplification caused by non-optimal PCR designs. We achieved the detection of 5 copies of M. tuberculosis genomic DNA (equaling 0.3 cell) in real biofluids using both optimized and non-optimal PCR designs, which is 10- and 1000-fold fewer than those of the standard bench-top method, respectively. By simplifying the workflow and shortening the development cycle of NAATs, our platform may find use in point-of-care diagnosis.     

Disposable on‐chip microfluidic system for buccal cell lysis,DNA purification,and polymerase chain reaction

This paper reports the development of a disposable, integrated biochip for DNA sample preparation and PCR. The hybrid biochip (25 × 45 mm) is composed of a disposable PDMS layer with a microchannel chamber and reusable glass substrate integrated with a microheater and thermal microsensor. Lysis, purification, and PCR can be performed sequentially on this microfluidic device. Cell lysis is achieved by heat and purification is performed by mechanical filtration. Passive check valves are integrated to enable sample preparation and PCR in a fixed sequence. Reactor temperature is needed to lysis and PCR reaction is controlled within ±1°C by PID controller of LabVIEW software. Buccal epithelial cell lysis, DNA purification, and SY158 gene PCR amplification were successfully performed on this novel chip. Our experiments confirm that the entire process, except the off‐chip gel electrophoresis, requires only approximately 1 h for completion. This disposable microfluidic chip for sample preparation and PCR can be easily united with other technologies to realize a fully integrated DNA chip.     

Functional integration of DNA purification and concentration into a real time micro-PCR chip

Microfluidic devices for on-chip amplification of DNA from various biological and environmental samples have gained extensive attention over the past decades with many applications including molecular diagnostics of disease, food safety and biological warfare testing. But the integration of sample preparation functions into the chip remains a major hurdle for practical application of the chip-based diagnostic system. We present a PCR-based molecular diagnostic device comprised of a microfabricated chip and a centrifugal force assisted liquid handling tube (CLHT) that is designed to carry out concentration and purification of DNA and subsequent amplification of the target gene in a single chip. The reaction chamber of the chip contains an array of pillar structures to increase the surface area for capturing DNA from a raw sample of macro volume in the presence of kosmotropic agents. The CLHT was designed to provide an effective interface between sample preparation and the microfluidic PCR chip. We have characterized the effect of various fluidic parameters including DNA capture, amplification efficiency and centrifugal pressure generated upon varying sample volume. We also evaluated the performance of this system for quantitative detection of E. coli O157:H7. From the samples containing 10(1) to 10(4) cells per mL, the C(T) value linearly increased from 25.1 to 34.8 with an R(2) value greater than 0.98. With the effectiveness and simplicity of operation, this system will provide an effective interface between macro and micro systems and bridge chip-based molecular diagnosis with practical applications.     

Under-three minute PCR: probing the limits of fast amplification

Nucleic acid amplification is enormously useful to the biotechnology and clinical diagnostic communities; however, to date point-of-use PCR has been hindered by thermal cycling architectures and protocols that do not allow for near-instantaneous results. In this work we demonstrate PCR amplification of synthetic SARS respiratory pathogenic targets and bacterial genomic DNA in less than three minutes in a hardware configuration utilizing convenient sample loading and disposal. Instead of sample miniaturization techniques, near-instantaneous heating and cooling of 5 μL reaction volumes is enabled by convective heat transfer of a thermal fluid through porous media combined with an integrated electrical heater. This method of rapid heat transfer has enabled 30 cycles of PCR amplification to be completed in as little as two minutes and eighteen seconds. Surprisingly, multiple enzymes have been shown to work at these breakthrough speeds on our system. A tool for measuring enzyme kinetics now exists and can allow polymerase optimization through directed evolution studies. Pairing this instrument technology with modified polymerases should result in a new paradigm for high-throughput, ultra-fast PCR and will hopefully improve our ability to quickly respond to the next viral pandemic.     

One‐Step Nucleic Acid Purification and Noise‐Resistant Polymerase Chain Reaction by Electrokinetic Concentration for Ultralow‐Abundance Nucleic Acid Detection

Nucleic acid amplification tests (NAATs)integrated on a chip hold great promise for point‐of‐care diagnostics. Currently, nucleic acid (NA) purification remains time‐consuming and labor‐intensive, and it takes extensive efforts to optimize the amplification chemistry. Using selective electrokinetic concentration, we report one‐step, liquid‐phase NA purification that is simpler and faster than conventional solid‐phase extraction. By further re‐concentrating NAs and performing polymerase chain reaction (PCR) in a microfluidic chamber, our platform suppresses non‐specific amplification caused by non‐optimal PCR designs. We achieved the detection of 5 copies of M. tuberculosis genomic DNA (equaling 0.3 cell) in real biofluids using both optimized and non‐optimal PCR designs, which is 10‐ and 1000‐fold fewer than those of the standard bench‐top method, respectively. By simplifying the workflow and shortening the development cycle of NAATs, our platform may find use in point‐of‐care diagnosis.     

Serial processing of biological reactions using flow-through microfluidic devices: coupled PCR/LDR for the detection of low-abundant DNA point mutations

We have fabricated a flow-through biochip consisting of passive elements for the analysis of single base mutations in genomic DNA using polycarbonate (PC) as the substrate. The biochip was configured to carry out two processing steps on the input sample, a primary polymerase chain reaction (PCR) followed by an allele-specific ligation detection reaction (LDR) for scoring the presence of low abundant point mutations in genomic DNA. The operation of the device was demonstrated by detecting single nucleotide polymorphisms in gene fragments (K-ras) that carry high diagnostic value for colorectal cancers. The effect of carryover from the primary PCR on the subsequent LDR was investigated in terms of LDR yield and fidelity. We found that a post-PCR treatment step prior to the LDR phase of the assay was not essential. As a consequence, a thermal cycling microchip was used for a sequential PCR/LDR in a simple continuous-flow format, in which the following three steps were carried out: (1) exponential amplification of the gene fragments from genomic DNA; (2) mixing of the resultant PCR product(s) with an LDR cocktail via a Y-shaped passive micromixer; and (3) ligation of two primers (discriminating primer that carried the complement base to the mutation locus being interrogated and a common primer) only when the particular mutation was present in the genomic DNA. We successfully demonstrated the ability to detect one mutant DNA in 1000 normal sequences with the integrated microfluidic system. The PCR/LDR assay using the microchip performed the entire assay at a relatively fast processing speed: 18.7 min for 30 rounds of PCR, 4.1 min for 13 rounds of LDR (total processing time = ca. 22.8 min) and could screen multiple mutations simultaneously in a multiplexed format. In addition, the low cost of the biochip due to the fact that it was fabricated from polymers using replication technologies and consisted of passive elements makes the platform amenable to clinical diagnostics, where one-time use devices are required to eliminate false positives resulting from carryover contamination.     

A Review of Multifunctions of Dielectrophoresis in Biosensors and Biochips for Bacteria Detection

This paper reviews the functions of dielectrophoresis (DEP) that have been applied to biosensor and biochip platforms for bacteria detection, including concentration of bacterial cells from continuous flows, separation of target bacterial cells from non-target cells, as well as the enhancement of antibody capture efficiency on biosensor and biochip surfaces. DEP could provide effective concentration and separation simultaneously in well-designed microfluidic biosensor and biochip systems. The integration of DEP with a detection system allows the integration of sample preparation and enrichment steps with detection, which has the potential to eliminate the traditionally used time-consuming culture-based enrichment steps and other multiple off-chip sample preparation steps. DEP is also useful in biosensor and biochips platforms for enhancing antibody capture efficiency in both flow-through and non-flow-through microdevices. The enhanced antibody capture efficiency could allow the sensor capture more cells and to be detected by the sensor, particularly in dealing with low number of cells. The integration of multifunctions of DEP into biosensor and biochip platform has the potential to improve the detection of bacterial cells.     

Solid phase DNA extraction on PDMS and direct amplification

In this paper we report an innovative use of Poly(DiMethyl)Siloxane (PDMS) to design a microfluidic device that combines, for the first time, in one single reaction chamber, DNA purification from a complex biological sample (blood) without elution and PCR without surface passivation agents. This result is achieved by exploiting the spontaneous chemical structure and nanomorphology of the material after casting. The observed surface organization leads to spontaneous DNA adsorption. This property allows on-chip complete protocols of purification of complex biological samples to be performed directly, starting from cells lysis. Amplification by PCR is performed directly on the adsorbed DNA, avoiding the elution process that is normally required by DNA purification protocols. The use of one single microfluidic volume for both DNA purification and amplification dramatically simplifies the structure of microfluidic devices for DNA preparation. X-Ray Photoelectron Spectroscopy (XPS) was used to analyze the surface chemical composition. Atomic Force Microscopy (AFM) and Field Emission Scanning Electron Microscopy (FESEM) were employed to assess the morphological nanostructure of the PDMS-chips. A confocal fluorescence analysis was utilized to check DNA distribution inside the chip.     

Supercoiled plasmid DNA purification by integrating membrane technology with a monolithic chromatography

The present study describes the integration of membrane technology with monolithic chromatography to obtain plasmid DNA with high quality. Isolation and clarification of plasmid DNA lysate were first conducted by a microfiltration step, by using a hydrophilic nylon microfiltration membrane, avoiding the need of centrifugation. For the total elimination of the remaining impurities, a suitable purification step is required. Monolithic stationary phases have been successfully applied as an alternative to conventional supports. Thus, the sample recovered from the membrane process was applied into a nongrafted CarbonylDiImidazole disk. Throughout the global procedure, a reduced level of impurities such as proteins and RNA was obtained, and no genomic DNA was detectable in the plasmid DNA sample. The chromatographic process demonstrated an efficient performance on supercoiled plasmid DNA purity and recovery (100 and 84.44%, respectively). Thereby, combining the membrane technology to eliminate some impurities from lysate sample with an efficient chromatographic strategy to purify the supercoiled plasmid DNA arises as a powerful approach for industrial‐scale systems aiming at plasmid DNA purification.     

An automated capillary electrophoresis system for high-speed separation of DNA fragments based on a short capillary

A high-speed DNA fragment separation system was developed based on a short capillary and a slotted-vial array automated sample introduction system. The injection process of DNA sample in a short capillary was investigated systematically with three injection techniques including constant-field-strength, low-field-strength and translational spontaneous injections. Under the optimized conditions, picoliter-scale sample plugs (corresponding to ca. 20-μm plug length) were obtained, which ensure the high-speed and high-efficiency separation for DNA fragments with a short effective separation length. Other separation conditions including the sieving matrix concentration, separation field strength and effective separation length were also optimized. The present system was applied in the separation of ΦX174-Hae III digest DNA marker. With an effective separation length of 2.5 cm, the separation could be achieved in <100 s with plate heights ranging from 0.21 to 0.74 μm (corresponding to plate numbers from 4.86 × 10(6) to 1.36 × 10(6)/m). The repeatabilities for the migration time of the eleven fragments were between 0.4 and 1.1% RSD (n=8). By using the automated continuous injection method, the separation for four different DNA samples could be achieved within 250 s. The present system was further applied in the fast sizing of real DNA samples of PCR products.     

Quantitative and qualitative analysis of a microfluidic DNA extraction system using a nanoporous AlO(x) membrane

A nanoporous aluminium oxide membrane was integrated into a microfluidic system designed to extract hgDNA (human genomic DNA) from lysed whole blood. The effectiveness of this extraction system was determined by passing known concentrations of purified hgDNA through nanoporous membranes with varying pore sizes and measuring the amount of hgDNA deposited on the membrane while also varying salt concentration in the solution. DNA extraction efficiency increased as the salt concentration increased and nanopore size decreased. Based on these results, hgDNA was extracted from whole blood while varying salt concentration, nanopore size and elution buffer to find the conditions that yield the maximum concentration of hgDNA. The optimal conditions were found to be using a low-salt lysis solution, 100 nm pores, and a cationic elution buffer. Under these conditions the combination of flow and ionic disruption were sufficient to elute the hgDNA from the membrane. The extracted hgDNA sample was analysed and evaluated using PCR (polymerase chain reaction) to determine whether the eluted sample contained PCR inhibition factors. Eluted samples from the microfluidic system were amplified without any inhibition effects. PCR using extracted samples was demonstrated for several genes of interest. This microfluidic DNA extraction system based on embedded membranes will reduce the time, space and reagents needed for DNA analysis in microfluidic systems and will prove valuable for sample preparation in lab-on-a-chip applications.     

MEMS-based sample preparation for molecular diagnostics

Completion of the Human Genome Project is driving the rapid development of molecular diagnostics in the laboratory. To accelerate the penetration of genetic tests and other nucleic acid-based tests into clinical markets, simple, compact, automatic sample-preparation systems for molecular diagnostics must be developed. Microelectromechanical systems (MEMS) is a promising approach for the development of automated sample preparation for the clinical laboratory or point-of-care setting. This review discusses MEMS-based components that could be applied to the different stages of the sample-preparation process such as cell separation, nucleic acid purification, and nucleic acid amplification. Examples of functional component integration are given. Issues discussed include partitioning of functions between the instrument and disposable unit, methods of propulsion of fluids and particles, vapor and liquid barriers, and sample size. Although further evaluation and development are needed to provide practical solutions to some of these issues, we conclude that MEMS-based components might contribute to some components in a sample-preparation system consisting of modular instruments and disposable units, but will not provide a generic or a totally integrated solution.     

Human genomic DNA analysis using a semi-automated sample preparation, amplification, and electrophoresis separation platform

The growing importance of analyzing the human genome to detect hereditary and infectious diseases associated with specific DNA sequences has motivated us to develop automated devices to integrate sample preparation, real-time PCR, and microchannel electrophoresis (MCE). In this report, we present results from an optimized compact system capable of processing a raw sample of blood, extracting the DNA, and performing a multiplexed PCR reaction. Finally, an innovative electrophoretic separation was performed on the post-PCR products using a unique MCE system. The sample preparation system extracted and lysed white blood cells (WBC) from whole blood, producing DNA of sufficient quantity and quality for a polymerase chain reaction (PCR). Separation of multiple amplicons was achieved in a microfabricated channel 30 microm x 100 microm in cross section and 85 mm in length filled with a replaceable methyl cellulose matrix operated under denaturing conditions at 50 degrees C. By incorporating fluorescent-labeled primers in the PCR, the amplicons were identified by a two-color (multiplexed) fluorescence detection system. Two base-pair resolution of single-stranded DNA (PCR products) was achieved. We believe that this integrated system provides a unique solution for DNA analysis.     

Genotyping from saliva with a one-step microdevice

This paper presents a disposable microfluidic device for on-chip lysing, PCR, and analysis in one continuous-flow process. Male-female sex determination was performed with human saliva in less than 20 min from spit to finish, and requiring only seconds of manual sample handling. This genetic analysis was based on the amplification and detection of the DYZ1 repeat region unique to the Y-chromosome. The flow-through microfluidic chip consisted of a single serpentine channel designed to guide samples through 42 heating and cooling cycles. Cycling was performed by matching the local channel geometry to a steady-state temperature gradient established across the microfluidic chip. 38 channel segments were designed for rapid low volume PCR, and four were optimized for spatial DNA melting analysis. Fluorescence detection was used to monitor the amplification and to capture the melting signature of the amplicon was performed with a basic 8-bit CCD camera. The microfluidic device itself was fabricated from microscope slides and a double-sided tape. The simplicity of the system and its robust performance combine in an elegant solution for lab-on-a-chip genetic analysis.