DNA Analysis Using an Integrated Microchip for Multiplex PCR Amplification and Electrophoresis for Reference Samples

• Published in: Analytical chemistry (Washington) 2014-08, Vol.86 (16), p.8192-8199
• Main Authors: Le Roux, Delphine, Root, Brian E, Reedy, Carmen R, Hickey, Jeffrey A, Scott, Orion N, Bienvenue, Joan M, Landers, James P, Chassagne, Luc, de Mazancourt, Philippe
• Format: Article
• Language: English
• Subjects: Amplification; Analytical chemistry; Chemical reactions; Chips; Deoxyribonucleic acid; DNA; DNA - analysis; Electrocatalysis; Electrophoresis, Microchip - instrumentation; Equipment Design; Forensic engineering; Humans; Ladders; Microfluidics; Multiplex Polymerase Chain Reaction - instrumentation; Multiplexing; Oligonucleotide Array Sequence Analysis - instrumentation; Polymerase chain reaction; Separation
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac501666b

A system that automatically performs the PCR amplification and microchip electrophoretic (ME) separation for rapid forensic short tandem repeat (STR) forensic profiling in a single disposable plastic chip is demonstrated. The microchip subassays were optimized to deliver results comparable to conventional benchtop methods. The microchip process was accomplished in sub-90 min compared with >2.5 h for the conventional approach. An infrared laser with a noncontact temperature sensing system was optimized for a 45 min PCR compared with the conventional 90 min amplification time. The separation conditions were optimized using LPA-co-dihexylacrylamide block copolymers specifically designed for microchip separations to achieve accurate DNA size calling in an effective length of 7 cm in a plastic microchip. This effective separation length is less than half of other reports for integrated STR analysis and allows a compact, inexpensive microchip design. This separation quality was maintained when integrated with microchip PCR. Thirty samples were analyzed conventionally and then compared with data generated by the microfluidic chip system. The microfluidic system allele calling was 100% concordant with the conventional process. This study also investigated allelic ladder consistency over time. The PCR-ME genetic profiles were analyzed using binning palettes generated from two sets of allelic ladders run three and six months apart. Using these binning palettes, no allele calling errors were detected in the 30 samples demonstrating that a microfluidic platform can be highly consistent over long periods of time.