Genomic DNA extraction from cells by electroporation on an integrated microfluidic platform.

The vast majority of genetic analysis of cells involves chemical lysis for release of DNA molecules. However, chemical reagents required in the lysis interfere with downstream molecular biology and often require removal after the step. Electrical lysis based on irreversible electroporation is a promising technique to prepare samples for genetic analysis due to its purely physical nature, fast speed, and simple operation. However, there has been no experimental confirmation on whether electrical lysis extracts genomic DNA from cells in a reproducible and efficient fashion in comparison to chemical lysis, especially for eukaryotic cells that have most of the DNA enclosed in the nucleus. In this work, we construct an integrated microfluidic chip that physically traps a low number of cells, lyses the cells using electrical pulses rapidly, then purifies and concentrates genomic DNA. We demonstrate that electrical lysis offers high efficiency for DNA extraction from both eukaryotic cells (up to ∼36% for Chinese hamster ovary cells) and bacterial cells (up to ∼45% for Salmonella typhimurium) that is comparable to the widely used chemical lysis. The DNA extraction efficiency has dependence on both the electric parameters and relative amount of beads used for DNA adsorption. We envision that electroporation-based DNA extraction will find use in ultrasensitive assays that benefit from minimal dilution and simple procedures.

[1]  Dino Di Carlo,et al.  On-chip cell lysis by local hydroxide generation. , 2005, Lab on a chip.

[2]  Stephen R. Quake,et al.  Microfluidic Digital PCR Enables Multigene Analysis of Individual Environmental Bacteria , 2006, Science.

[3]  Chang Lu,et al.  A microfluidic flow-through device for high throughput electrical lysis of bacterial cells based on continuous dc voltage. , 2006, Biosensors & bioelectronics.

[4]  U. Zimmermann,et al.  Electric Field Pulses Can Induce Apoptosis , 1999, The Journal of Membrane Biology.

[5]  Jerome P Ferrance,et al.  A simple, valveless microfluidic sample preparation device for extraction and amplification of DNA from nanoliter-volume samples. , 2006, Analytical chemistry.

[6]  Victor M Ugaz,et al.  A pocket-sized convective PCR thermocycler. , 2007, Angewandte Chemie.

[7]  C. Batt,et al.  Nucleic acid purification using microfabricated silicon structures. , 2003, Biosensors & bioelectronics.

[8]  R. Wilson,et al.  Complete genome sequence of Salmonella enterica serovar Typhimurium LT2 , 2001, Nature.

[9]  Paul Vulto,et al.  A microfluidic approach for high efficiency extraction of low molecular weight RNA. , 2010, Lab on a chip.

[10]  Hiroyuki Noji,et al.  Sequential processing from cell lysis to protein assay on a chip enabling the optimization of an F(1)-ATPase single molecule assay condition. , 2009, Lab on a chip.

[11]  Stephen R Quake,et al.  Whole-genome molecular haplotyping of single cells , 2011, Nature Biotechnology.

[12]  S. Quake,et al.  Monolithic microfabricated valves and pumps by multilayer soft lithography. , 2000, Science.

[13]  Mark Bachman,et al.  Fast electrical lysis of cells for capillary electrophoresis. , 2003, Analytical chemistry.

[14]  T. Geng,et al.  Observing single cell NF-κB dynamics under stimulant concentration gradient. , 2012, Analytical chemistry.

[15]  Vincent Studer,et al.  A nanoliter-scale nucleic acid processor with parallel architecture , 2004, Nature Biotechnology.

[16]  D. Kent,et al.  High-throughput analysis of single hematopoietic stem cell proliferation in microfluidic cell culture arrays , 2011, Nature Methods.

[17]  Zhao-Lun Fang,et al.  Integration of single cell injection, cell lysis, separation and detection of intracellular constituents on a microfluidic chip. , 2004, Lab on a chip.

[18]  Yiqiong Zhao,et al.  Using polarization-shaped optical vortex traps for single-cell nanosurgery. , 2007, Nano letters.

[19]  J. Weaver,et al.  Theory of electroporation: A review , 1996 .

[20]  F. Vitzthum,et al.  Amplifiable DNA from gram-negative and gram-positive bacteria by a low strength pulsed electric field method. , 2000, Nucleic acids research.

[21]  Yung-Chiang Chung,et al.  Microfluidic chip for high efficiency DNA extraction. , 2004, Lab on a chip.

[22]  Roland Zengerle,et al.  Continuous microfluidic DNA extraction using phase-transfer magnetophoresis. , 2010, Lab on a chip.

[23]  Kelvin H. Lee,et al.  The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line , 2011, Nature Biotechnology.

[24]  Jerome P Ferrance,et al.  Chitosan as a polymer for pH-induced DNA capture in a totally aqueous system. , 2006, Analytical chemistry.

[25]  Chang Lu,et al.  High‐throughput and real‐time study of single cell electroporation using microfluidics: Effects of medium osmolarity , 2006, Biotechnology and bioengineering.

[26]  Chang Lu,et al.  A microfluidic device for physical trapping and electrical lysis of bacterial cells , 2008 .

[27]  K. Jensen,et al.  A microfluidic electroporation device for cell lysis. , 2005, Lab on a chip.

[28]  R. Mathies,et al.  Integrated microfluidic systems for high-performance genetic analysis. , 2009, Trends in biotechnology.

[29]  Da Xing,et al.  Miniaturized PCR chips for nucleic acid amplification and analysis: latest advances and future trends , 2007, Nucleic acids research.

[30]  Chang Lu,et al.  One-step extraction of subcellular proteins from eukaryotic cells. , 2010, Lab on a chip.

[31]  Andreas Manz,et al.  Total nucleic acid analysis integrated on microfluidic devices. , 2007, Lab on a chip.

[32]  Xing Chen,et al.  On‐line cell lysis and DNA extraction on a microfluidic biochip fabricated by microelectromechanical system technology , 2008, Electrophoresis.

[33]  Da Xing,et al.  Single-molecule DNA amplification and analysis using microfluidics. , 2010, Chemical reviews.

[34]  Jerome P Ferrance,et al.  Microchip‐Based Cell Lysis and DNA Extraction from Sperm Cells for Application to Forensic Analysis , 2006, Journal of forensic sciences.

[35]  C. Klapperich,et al.  Cell lysis and DNA extraction of gram-positive and gram-negative bacteria from whole blood in a disposable microfluidic chip. , 2009, Lab on a chip.

[36]  M. Altwegg,et al.  A simple "universal" DNA extraction procedure using SDS and proteinase K is compatible with direct PCR amplification. , 1995, PCR methods and applications.

[37]  M. McClain,et al.  Microfluidic devices for the high-throughput chemical analysis of cells. , 2003, Analytical chemistry.

[38]  M. Heller,et al.  Preparation and hybridization analysis of DNA/RNA from E. coli on microfabricated bioelectronic chips , 1998, Nature Biotechnology.

[39]  Jeong-Woo Choi,et al.  Electrochemical cell lysis device for DNA extraction. , 2010, Lab on a chip.

[40]  Bo Yu,et al.  Nanochannel electroporation delivers precise amounts of biomolecules into living cells. , 2011, Nature nanotechnology.

[41]  Anubhav Tripathi,et al.  Microfluidic reactors for diagnostics applications. , 2011, Annual review of biomedical engineering.

[42]  Brian N. Johnson,et al.  An integrated microfluidic device for influenza and other genetic analyses. , 2005, Lab on a chip.

[43]  D. Chiu,et al.  Selective encapsulation of single cells and subcellular organelles into picoliter- and femtoliter-volume droplets. , 2005, Analytical chemistry.

[44]  Daniel C Leslie,et al.  Nucleic acid extraction techniques and application to the microchip. , 2009, Lab on a chip.

[45]  Michael G. Roper,et al.  A fully integrated microfluidic genetic analysis system with sample-in–answer-out capability , 2006, Proceedings of the National Academy of Sciences.

[46]  Samuel Aparicio,et al.  High-throughput microfluidic single-cell RT-qPCR , 2011, Proceedings of the National Academy of Sciences.

[47]  Kristen L. Helton,et al.  Microfluidic Overview of Global Health Issues Microfluidic Diagnostic Technologies for Global Public Health , 2006 .

[48]  Hsin-Chih Yeh,et al.  Single-molecule detection and probe strategies for rapid and ultrasensitive genomic detection. , 2005, Current pharmaceutical biotechnology.

[49]  Jungkyu Kim,et al.  Microfluidic sample preparation: cell lysis and nucleic acid purification. , 2009, Integrative biology : quantitative biosciences from nano to macro.

[50]  Brian E. Henslee,et al.  Electroporation dependence on cell size: optical tweezers study. , 2011, Analytical chemistry.

[51]  Chang Lu,et al.  Microfluidic chemical cytometry based on modulation of local field strength. , 2006, Chemical communications.

[52]  T. Geng,et al.  Histone modification analysis by chromatin immunoprecipitation from a low number of cells on a microfluidic platform. , 2011, Lab on a chip.

[53]  Yusi Fu,et al.  Digital polymerase chain reaction in an array of femtoliter polydimethylsiloxane microreactors. , 2012, Analytical chemistry.

[54]  Chang Lu,et al.  Electroporation of mammalian cells in a microfluidic channel with geometric variation. , 2006, Analytical chemistry.

[55]  N. Munce,et al.  Microfabricated system for parallel single-cell capillary electrophoresis. , 2004, Analytical chemistry.

[56]  Piero R Bianco,et al.  Laminar flow cells for single-molecule studies of DNA-protein interactions , 2008, Nature Methods.

[57]  Stephen R Quake,et al.  Microfluidic single-cell mRNA isolation and analysis. , 2006, Analytical chemistry.

[58]  Juan G Santiago,et al.  Purification of nucleic acids from whole blood using isotachophoresis. , 2009, Analytical chemistry.

[59]  Anupam Singhal,et al.  Megapixel digital PCR , 2011, Nature Methods.

[60]  Stephen R Quake,et al.  Genomic analysis at the single-cell level. , 2011, Annual review of genetics.

[61]  Bruce K Gale,et al.  Quantitative and qualitative analysis of a microfluidic DNA extraction system using a nanoporous AlO(x) membrane. , 2008, Lab on a chip.