Integrated Portable Polymerase Chain Reaction-Capillary Electrophoresis Microsystem for Rapid Forensic Short Tandem Repeat Typing

Abstract

A portable forensic genetic analysis system consisting of a microfluidic device for amplification and separation of short tandem repeat (STR) fragments as well as an instrument for chip operation and four-color fluorescence detection has been developed. The microdevice performs polymerase chain reaction (PCR) in a 160-nL chamber and capillary electrophoresis (CE) in a 7-cm-long separation channel. The instrumental design integrates PCR thermal cycling, electrophoretic separation, pneumatic valve fluidic control, and four-color laser excited fluorescence detection. A quadruplex Y-chromosome STR typing system consisting of amelogenin and three Y STR loci (DYS390, DYS393, DYS439) was developed and used for validation studies. The multiplex amplification of these 4 loci with 35 PCR cycles followed by CE separation and 4-color fluorescence detection was completed in 1.5 h. All the amplicons can be detected with a limit of detection of 20 copies of male standard DNA in the reactor. Real-world forensic analyses of oral swab and human bone extracts from case evidence were also successfully performed. Mixture analysis demonstrated that a balanced profile can be obtained even at a male-to-female template ratio of 1:10. The successful development and operation of this portable PCR-CE system establishes the feasibility of rapid point-of-analysis DNA typing of forensic casework, of mass disaster samples or of individuals at a security checkpoint.

Microwave Dielectric Heating of Fluids in Microfluidic Devices

“…The data also resulted in a quasilinear temperature gradient with a slope of 7.3 °C mm −1 along a 2 mm distance. Linear temperature gradients with comparable slopes have been used for DNA mutation detection (Bienvenue et al, 2006), phase transition measurements in phospholipid membranes (Saiki et al, 1986), singlenucleotide polymorphism (SNP) analysis (Baker., 1995) and continuous-flow thermal gradient PCR (Liu et al, 2007). As before, nonlinear least-squares fitting of the theoretical model (solid line in Figure 16) to the experimental data shows good agreement (R 2 = 0.98).…”

Recent Progress of On-line Combination of Preconcentration Device with Microchip Electrophoresis

“…In a microfluidic device of which volume of microchambers/channels is extremely small, temperature of a sample solution can be varied easily and rapidly. Thus, a further high-throughput PCR with a shorter amplification time can be employed as compared to commercialized showed good resolutions and similar signal to noise ratio as compared to those obtained by previous reports [58]. These integrated microsystems will be a significant advance in the development of high-throughput and highly sensitive µTAS for on-site DNA analysis.…”

Optically addressable single-use microfluidic valves by laser printer lithography

“…[10][11][12][13] There have been a number of demonstrations of microfluidic systems in thermoplastics, 10,11,14,15 but these examples are a rarity compared to microfluidic devices based on PDMS and glass. 2,4,6,9,[16][17][18] Arguably, integrated microfluidic devices based on PDMS and glass [2][3][4]6,7,9 could be reconfigured as thermoplastic devices, but to date the examples of this are few, and researchers continue to actively seek ways to substitute PDMS for thermoplastics. [19][20][21][22] Our belief is that integrated microfluidic systems should be designed from the outset with consideration of batch fabrication, volume manufacture, and reproducibility, and that this naturally leads to a preference for thermoplastic components and structures.…”