Automated parallel DNA sequencing on multiple channel microchips

Abstract

We report automated DNA sequencing in 16-channel microchips. A microchip prefilled with sieving matrix is aligned on a heating plate affixed to a movable platform. Samples are loaded into sample reservoirs by using an eight-tip pipetting device, and the chip is docked with an array of electrodes in the focal plane of a four-color scanning detection system. Under computer control, high voltage is applied to the appropriate reservoirs in a programmed sequence that injects and separates the DNA samples. An integrated fourcolor confocal fluorescent detector automatically scans all 16 channels. The system routinely yields more than 450 bases in 15 min in all 16 channels. In the best case using an automated base-calling program, 543 bases have been called at an accuracy of >99%. Separations, including automated chip loading and sample injection, normally are completed in less than 18 min. The advantages of DNA sequencing on capillary electrophoresis chips include uniform signal intensity and tolerance of high DNA template concentration. To understand the fundamentals of these unique features we developed a theoretical treatment of cross-channel chip injection that we call the differential concentration effect. We present experimental evidence consistent with the predictions of the theory.

Surface modification in microchip electrophoresis

“…CE/MS analysis of a four-protein mixture using a PAA-coated microchip coupled to an ESI-MS. The sample concentration was 2 mM of each protein in 5 mM NH 4 Ac/ HAc (pH 4.4). Reprinted from [74], with permission.…”

“…Microfluidic devices have been applied for electrophoretic separations for a variety of biochemical and chemical analysis [1][2][3][4][5][6][7]. Latest developments in microchip electrophoresis (MCE) have been summarized in recent reviews [8][9][10][11][12][13].…”

Rapid Enantioseparation of 1-Aminoindan by Microchip Electrophoresis with Linear-Imaging UV Detection

“…Microchip electrophoresis (MCE), based on the separation principle of CE, has been widely used in the analysis of chemical and biogenic compounds because of its high performance, high speed, compactness, and low consumption of solvents and samples. [14][15][16][17][18][19][20] These characteristics of MCE are very suitable for the development of a high-throughput chiral-analysis system.…”

Ultrafast DNA sequencing on a microchip by a hybrid separation mechanism that gives 600 bases in 6.5 minutes

“…Since then, many other papers reporting four-color DNA sequencing in glass and plastic chips have been published (10,(20)(21)(22)(23)(24), with Salas-Solano et al (19) achieving 580 base reads in 18 min and 640 base reads in 30 min in single-channel fused silica glass chips. In these studies, read lengths are reported either with a certain accuracy when obtained read lengths are compared with the known DNA sequence (usually using a cutoff accuracy of 98.5%) or by using a quality assessment program called phred, which assigns a quality score based on peak shapes, where a called base with a phred score of 20 (Q20) has a 99% probability of being correct.…”

“…In these studies, read lengths are reported either with a certain accuracy when obtained read lengths are compared with the known DNA sequence (usually using a cutoff accuracy of 98.5%) or by using a quality assessment program called phred, which assigns a quality score based on peak shapes, where a called base with a phred score of 20 (Q20) has a 99% probability of being correct. To demonstrate the potential for increased throughput of microfluidic sequencing devices, Liu et al (20) Here, we report a microfluidic chip-based system that provides ultrafast DNA sequencing, providing read lengths up to 600 bases in 6.5 min using a high-performance polymer system comprised of both a separation matrix and a physically adsorbed microchannel wall coating. Previously published microchip sequencing results have exclusively used linear polyacrylamide (LPA) as the sequencing matrix as well as a covalently bonded LPA coating for the channel walls, with analysis times in these systems ranging from 15 to 35 min.…”