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

Abstract

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.

The development of miniSTRs as a method for high‐speed direct PCR

“…For a fast presumptive DNA analysis method that can be used when time is limited, the developed rapid direct two-step PCR method should be combined with high-speed microfluidic separation and detection methods. Previous studies have shown the use of microchips for separation and detection of DNA fragments [22,23,[41][42][43]. For example, Anazawa et al developed a multi-channel microchip electrophoresis system with five-color and eight-channel fluorescence detection.…”

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

“…The detailed information on this instrument can be found in our previous study. 10,31 Modification of Quartz Filter Paper with Chitosan. Chitosan (low molecular weight), MES (2-(N-morpholino) ethanesulfonic acid), SDS (sodium dodecyl sulfonate), and GPTMS ((3-glycidyloxypropyl) trimethoxysilane) were all purchased from Sigma-Aldrich (St. Louis, MO).…”

“…6−10 For example, Le Roux et al reported a microfluidic system that integrated DNA extraction, PCR, and capillary electrophoresis together on a single multilayer plastic device. 8 Our group also developed a fully integrated microsystem for STR typing within 2 h. 10 Commercialized systems, such as RapidHIT (IntegenX, Pleasanton, CA) and DNAScan (GE Healthcare, Pittsburgh, PA), 11,12 have been deployed to forensic laboratories for extensive validation. This progress announces the era of automatically conducting STR analysis whenever and wherever needed is approaching.…”

“…In these situations, high throughput may not be required, but the automation and the portability are highly desired. To fulfill this need, several groups including us have reported the successful development of integrated microfluidic devices, which could generate STR profiles in a “sample-in-answer-out” manner. − For example, Le Roux et al reported a microfluidic system that integrated DNA extraction, PCR, and capillary electrophoresis together on a single multilayer plastic device . Our group also developed a fully integrated microsystem for STR typing within 2 h .…”

“…To fulfill this need, several groups including us have reported the successful development of integrated microfluidic devices, which could generate STR profiles in a “sample-in-answer-out” manner. − For example, Le Roux et al reported a microfluidic system that integrated DNA extraction, PCR, and capillary electrophoresis together on a single multilayer plastic device . Our group also developed a fully integrated microsystem for STR typing within 2 h . Commercialized systems, such as RapidHIT (IntegenX, Pleasanton, CA) and DNAScan (GE Healthcare, Pittsburgh, PA), , have been deployed to forensic laboratories for extensive validation.…”