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

Abstract

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.

Microchip capillary electrophoresis with a cellulose-DNA-modified screen-printed electrode for the analysis of neurotransmitters

“…The sharper peaks were obtained at the modified electrode; in contrast, the broader peaks were resulted at the bare electrode. At the modified electrode, the resolution (R), which was calculated following a previously described equation [31], was found to be much better than that of the bare one. The R value between the DA and the NE at the modified electrode was found to be 2.2, whereas at the bare one, it was [17,18,27].…”

Together with 30 years of Symposia on Chrysomelidae! Memories and personal reflections on what we know more about leaf beetles

“…Let us quote some of them: metafemoral spring of flea beetles and jumping by David Furth (1988, Furth and Suzuki 1994, 1998), and Michael Schmitt (2004); meioformulae of Leaf Beetles by Petitpierre (1997, 1999, 2011), Virkki (1985, 1988, 1989) and others; larvae research, by Steinhausen (1985, 1994, 1995, 1996) and others; chemical defense by Jacques Pasteels (Pasteels and Hartmann 2004, Pasteels and Rowell-Rahier 1989, Pasteels et al 1986, 1988, 1989, 1994, 1996, 2004); cycloalexy, by Vasconcellos-Neto and Jolivet (1989, 1994); fossils by Santiago-Blay (1994; Santiago-Blay and Craig 1999, Santiago-Blay et al 1996, 2004), followed by many others; mimicry by Balsbaugh (1988, Balsbaugh and Fauske 1991) and many others; zoogeography by Verma (Verma and Jolivet 2004, 2006), Scherer (1988), Daccordi (1994, 1996, 2000, 2003a, b, c), and many more; egg bursters by Cox (1988, 1994); structure of ovaries and viviparity by Christian Bontems (1988, Bontemps and Lee 2008); Criocerinae biology, by Fredric Vencl (Vencl and Morton 1998, 1999, Vencl and Nishida 2008, Vencl et al 2004), M. Schmitt (1988), Yoko Matsumura (Matsumura and Akimoto 2009, Matsumura and Suzuki 2008, Matsumura and Yoshizawa 2010, 2012, Matsumura et al 2010, 2012); African fauna of Alticinae by Maurizio Biondi (1989, 1999, 2001a, b, Biondi and D’Alessandro 2008, 2010a, b, 2012); Australian fauna by Mauro Daccordi (2000, 2003a, b, c, Daccordi and DeLittle 2003), Chris Reid (1989, 1991a, b, 1992, 2003, 2006, Reid and Beat...…”

Clinical Analysis by Microchip Capillary Electrophoresis

“…Microchip CE has been used for the analysis of genetic diseases such as Duchenne muscular dystrophy (DMD) (72)(73)(74) and hemochromatosis (75 ), and for genomic DNA analysis (76 ). DMD is caused by mutations in the dystrophin gene on the X chromosome.…”

“…A semiautomated sample preparation, amplification, and electrophoretic separation platform has also been developed for analysis of human genomic DNA to detect hereditary and infectious diseases based on microchannel CE with 2-color optical detection (excitation wavelengths at 488 and 532 nm). Two-base pair resolution of single-stranded DNA was achieved in the analysis of PCR products from leukocyte lysates (76 ).…”

“…Potential advantages of microchip CE include miniaturization, integration, high speed, and reduced reagent consumption. We present an overview of the approaches and selected applications of microchip CE devices in the diagnosis of major diseases, including cancer (33-41 ); cardiovascular (42-45 ), renal (46, 47 ), neurologic (48 -50 ), thyroid (51, 52 ), and infectious diseases (53-64 ); immune disorders (65-69 ); diabetes (70 -72 ); and hereditary diseases (73)(74)(75)(76)(77). We also briefly…”