1 / 11

A Nanoliter-Scale Nucleic Acid Processor with Parallel Architecture

A Nanoliter-Scale Nucleic Acid Processor with Parallel Architecture. Jong Wook Hong, Vincent Studer, Giao Hang, W French Andreson, Stephen R Quake. presented by: Anna Shcherbina Michael Meyer. Motivation for Single-Cell mRNA/ DNA Extraction. Goal : Use Single Cell To Establish

stew
Download Presentation

A Nanoliter-Scale Nucleic Acid Processor with Parallel Architecture

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. A Nanoliter-Scale Nucleic Acid Processor with Parallel Architecture Jong Wook Hong, Vincent Studer, Giao Hang, W French Andreson, Stephen R Quake presented by: Anna Shcherbina Michael Meyer

  2. Motivation for Single-Cell mRNA/ DNA Extraction • Goal: Use Single Cell To Establish • cDNA Library • Gene Expression Profile Isolating cells from animals or patients results in a mixture of cell types. Epigenetic variation between cells with identical genotypes influences development. Primary cells hard to obtain in large quantities .

  3. Existing Technologies and their Limitations • Affinity capture and elution of purified DNA from silicon microstructures • Deep Reactive Ion Etching (DRIE) • On the order of uL, not nL • No parallelization • No integration • Microarrays measure expression of a few genes from a single cell • Amplification process introduces distortion • Require choice of finite set of possible transcripts • Currently, cDNA library construction methods requires 1000-10,000 input cells.

  4. Innovation: Microfluidic Chip to Sequentially Process nL Volumes and Isolate Cells • Small-Volume Scaling • Process Integration • Fabricated by multi-layer soft lithography • Lysing and purification performed directly on the chip. • No pre/post-treatment needed. • Compatible with many biological assays • Protein crystallization • nL -volume PCR • FACS • single-cell enzyme screening

  5. mRNA Purification Chip Design affinity column bead chamber Lysing buffer chamber cell chamber b. Photograph of the in situ affinity column construction. Scale bar 200 um. c. Cell loaded into cell chamber before lysis step. Scale bar 100 um. (Hong et al.) Fig 1. a. Layout of microfluidic chip, version 1. Channels are 100 um wide. Fluidic ports are named; actuation ports are numbered 1-11.

  6. Performance & Sensitivity Assay • Primers used to identify high abundance B-actin transcript and moderate abundance OZF transcript. • 18 experiments performed. • 5 of these used a single cell. Fig 2. RT-PCR analysis of isolated mRNA. RT-PCR products analyzed on 2% agarose gel loaded with 5% of reaction. (Hong et al.)

  7. Chip Sensitivity Assay Results • B-actin purified from cells in 14 out of 18 experiments • Between 2-10 cells required to detect OZF mRNA signal • Monotonically increasing relationship between band intensity and cell number in B-actin mRNA. • No functional relationship observed in non-normalized data. Fig 3. RT-PCR products for both transcripts were analyzed on a 2% agarose gel, whose bands were quantified and normalized. Zero values indicate absence of detectable band in gel. (Hong et al.)

  8. DNA Purification Chip Architecture (advances) • Parallelization • Align several linear processors and use same cross-junction structures to load them simultaneously. • Loading & Processing Flow • Loading--fluid flows north/south • Processing--fluid flows east/west, along each batch processor • Customization Fig 4. Temporal action of DNA isolation circuitry (Hong et al.)

  9. Full Chip and Experimental Setup - Characterization of Sensitivity • Each processor hold 5 nL • Volume of cells used: 1.6, 1.0, 0.4 nL • remaining volume is reaction buffer and lysis buffer Fig 5. Food-coloring reveals the interconnectivity of chip. (Hong et al.)

  10. DNA Yield Experimental Results a. Undiluted E. coli culture Lane 1: 1.6 nL culture (~1120 cells) Lane 2: 1.0 nL culture (~700 cells) Lane 3: 0.4 nL culture (~280 cells) Lanes 4-6: Negative control (pure H2O) b. 1:10 dilutions Lanes 1,4,7: diluted 1.6 nL Lanes 2,5,8: diluted 1.0 nL Lanes 3,6,9: diltued 0.4 nL c. Intensity of gel bands Fig 6. Verification of the successful recovery of E. coli genomic DNA. Samples have been PCR amplified. (Hong et al.)

  11. Potential Uses and Impact • Increasing throughput in single-cell analysis • Automation of reagent preparation for large cell populations • industrial-scale microarray analysis • Preparation step for environmental analysis or medical diagnostics • Tool for microculture and analysis of slow-growing or unculturable bacteria • Generation of subtractive libraries from pairs of single cells • eliminate commonly expressed transcripts & • enrich differentially expressed transcripts

More Related