Exploiting biased reptation for continuous flow preparative DNA fractionation in a versatile microfluidic platform

Abstract

A new approach is presented for preparative, continuous flow fractionation of sub-10-kbp DNA fragments, which exploits the variation in the field-dependent mobility of the DNA molecules based on their length. Orthogonally pulsed electric fields of significantly different magnitudes are applied to a microchip filled with a sieving matrix of 1.2% agarose gel. Using this method, we demonstrate a high-resolution separation of 0.5, 1, 2, 5, and 10 kbp DNA fragments within 2 min. During the separation, DNA fragments are also purified from other ionic species. Preparative fractionation of sub-10-kbp DNA molecules plays an important role in second-generation sequencing. The presented device performs rapid high-resolution fractionation and it can be reliably manufactured with simple microfabrication procedures. This method has the advantages of simplicity, versatility, and reproducibility. However, it suffers from long processing times on the order of a few tens of hours 2,3 . For instance, the Bio-Rad CHEF-DR device performs fractionation of 5-120 kbp DNA using pulsed-field gel electrophoresis (PFGE) in 25 h (Refs. 4,5). Trends toward second-generation sequencing motivate the replacement of standard gel electrophoresis with microchip-based systems, which could provide efficient platforms to minimize the processing time and to perform optimal DNA fractionation 6-8 .Micro-and nanofabricated post arrays-that resemble a gel matrix with well-ordered and identical pores-have been integrated in microchip-based systems, increasing both the understanding of DNA separation principles and the fractionation's efficiency and speed. Two decades ago, Volkmuth and Austin 9 introduced a patterned micro-post array in a microfluidic electrophoresis platform, in which DNA fragments were separated by biased reptation under a DC electric field. Later, Duke et al. separated large fragments in a range of 60-135 kbp in a microfabricated array using DNA reorientation-or the 'switchback' principle. Although the switchback principle is similar to what is used in PFGE, the device yields much faster separations owing to its sparse and regular sieving array 10 . In a later work, Kaji et al. studied a three-dimensional (3D) nanopost array as an optimal separation matrix for DNA fragments over a few kbps under a DC electric field, resulting in similarly fast separations due to the sparse matrix 11 .With the aim of increasing the sample throughput and further facilitating the sample recovery, a continuous flow separation was developed. Huang et al. 12 performed continuous flow PFGE in a micromachined post array by applying pulsed electric fields of slightly unequal strengths. DNA fragments ranging between 61 and 209 kbp were separated within 15 s in the 'DNA prism'.

Block Copolymer of Flexible and Semi-Flexible Block Confined in Nanopost Array

“…Recent advances in nanofabrication [4] have resulted in preparation of arrays of nanoposts that can be used for DNA separations or as templates for the block copolymer self-assemblies. A nanopost array appears as a possible replacement of a random gel matrix in gel electrophoresis [5,6]. The ability to control the polymer conformation in such regular nanostructures has led to controlled storage or retrieval of genetic information [7], the idea that stems from a comparison to complex natural organization of a DNA in the chromatin.…”

Microfluidic Desalination: A New Era Towards Sustainable Water Resources

“…Electrophoresis (EP) was initially used to fractionate DNA molecules based on their length. Thus, this method was implemented in police forensics as well as in genetic research 76. This phenomenon is related closely to the EO phenomenon discussed in the previous section.…”

Review on microfluidic device applications for fluids separation and water treatment processes

“…Gumuscu et al [31] developed a low cost and easy manufacturing microdevice made of glass, for ultrafast separation and purification of DNA. The new chip was capable to perform DNA fragmentation in only few minutes, while conventional approaches could take hours.…”

“…Microdevice design used to separate and fragmentate DNA samples: a glass microdevice, b microchip layout (reservoirs indicated), c support made of Delrin and its schematics. Reprinted from[31]…”