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

Abstract

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.

Ultrafast photonic PCR

“…Resistive heating with microfabricated thin film heaters is most commonly used to control the temperature in the static microfluidic-based PCR system, in which the PCR runs in the microfluidic chamber. 11,12 However, this method requires a complicated fabrication process to integrate the thin film heater and resistance temperature detection sensor on the chip. In the case of continuous-flow PCR on a chip, the PCR amplification occurs when the reaction samples pass thorough three discrete temperature zones.…”

“…Because fast/ultrafast PCR is highly desirable for applications such as timely diagnosis of infectious diseases, cardiac diseases, cancer, neurological disorder diseases, and rapid biowarfare and pathogen identification at the point-of-care (POC) level, many academic and industrial groups have worked on improving PCR systems, [9][10][11][12][13][14][15] One commercial PCR system (LightCycler H 2.0, Roche Diagnostics USA, Indianapolis, IN, USA) using air heating/cooling and capillary tubes can perform 30 thermal cycles in 10-60 min, depending on sample volume. 10 However, this system is not suitable for POC testing due to its high power consumption (800 W maximum) and heavy weight (approximately 22 kg).…”

Development of a PCR/LDR/Flow-Through Hybridization Assay Using a Capillary Tube, Probe DNA-Immobilized Magnetic Beads and Chemiluminescence Detection

“…[24][25][26][27] The use of beads as solid supports for capture probes for flow-through hybridization analysis is advantageous for the following reasons: (1) enhanced hybridization efficiency due to the effective mixing of solutions 23 and a significantly larger surface area compared with the interior surface area of a typical microfluidic channel; 22,23,28 (2) easier modification of the bead surfaces compared with the walls of a microfluidic path; 29,30 (3) facile positioning of beads in the microfluidic network using a microfabricated bottleneck, 20 weir, 16,19,22 and cage, 31 or more simply, one can place magnetic beads anywhere in the microfluidic network using small magnets. 18,21 Because the chemiluminescence (CL) detection method can provide high sensitivity while using simple instrumentation, it has become a powerful and essential tool for a wide range of applications in fields such as molecular biotechnology, pharmacology, and biomedical chemistry. [32][33][34][35] To the best of our knowledge, CL detection has not yet been reported for PCR/LDR/hybridization assays.…”

“…However, there are a number of studies in which flow-through hybridization of DNA onto probe-conjugated microbeads was successfully performed while being confined to microfluidic chambers [16][17][18][19][20][21][22][23] and capillary tubes. [24][25][26][27] The use of beads as solid supports for capture probes for flow-through hybridization analysis is advantageous for the following reasons: (1) enhanced hybridization efficiency due to the effective mixing of solutions 23 and a significantly larger surface area compared with the interior surface area of a typical microfluidic channel; 22,23,28 (2) easier modification of the bead surfaces compared with the walls of a microfluidic path; 29,30 (3) facile positioning of beads in the microfluidic network using a microfabricated bottleneck, 20 weir, 16,19,22 and cage, 31 or more simply, one can place magnetic beads anywhere in the microfluidic network using small magnets.…”

DNA capture-probe based separation of double-stranded polymerase chain reaction amplification products in poly(dimethylsiloxane) microfluidic channels

“…[8][9][10][11][12] Many strategies are based on a common technique which involves both the concentration of PCR products and separation from unreacted primers, nucleotide triphosphates, and dyes. In these cases, linear or crosslinked acrylamide hydrogel plugs located between the sample and waste channels in a classic double T-junction CE microfluidic system 13 are used.…”