[Illinois] ECE 416 DNA Microarrays I

By Brian Cunningham

University of Illinois at Urbana-Champaign

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Abstract

           In this lecture, we started off with a revision of DNA with genes and gene expressions and how DNA sends messages. The problem with gene detection is how to analyze expressions with thousands of genes and determine what possible physiological processes a gene may be related to. All cells have the same genome, but each cell can express different set of proteins. The mRNA sequences present in a cell can be seen with a DNA Microarray. On a surface, you can print thousands of individual gene sequences applied in a grid of "spots" to surface of a glass slide or a biosensor. There are two main types of arrays: long and short. But in both of them if a sequence is presented a fluorescent tag is attached and then excited with a laser so we can observe them. In long strand DNA Microarrays, the portion of coding region of gene to be analyzed is individually amplified by PCR. The DNA is covalently bound to the surface and the attached DNA is denatured leaving a single-strand DNA molecule attached. There are different DNA Array spot making methods, using pin-based or ink jet. It is difficult to make perfect spots all the time, so some are replicated in different areas so the results are not ruined by a "bad" spot. The short strand DNA Microarray method uses a "light detected synthesis" method and several nucleotide sequences from a single gene are synthesized in neighboring regions. In order to make these microarrays, each gene is built one base pair at a time and how they are made is discussed in further detail. For detection, an array of immobilized single-strand DNA "probes" is used to capture fluorescently tagged DNA targets from the test sample. The probe-target binding is only strong when base pairs are exactly complementary to each other. The fluorescent tags are attached to the detected (analyte) DNA and the ratio of the colors of the sample to the control is used to figure out detection of the molecules.

Bio

My research group is focused on the application of sub-wavelength optical phenomena and fabrication methods to the development of novel devices and instrumentation for the life sciences. The group is highly interdisciplinary, with expertise in the areas of microfabrication, nanotechnology, computer simulation, instrumentation, molecular biology, and cell biology. In particular, we are working on biosensors based upon photonic crystal concepts that can either be built from low-cost flexible plastic materials, or integrated with semiconductor-based active devices, such as light sources and photodetectors, for high performance integrated detection systems.

Using a combination of micrometer-scale and nanometer-scale fabrication tools, we are devising novel methods and materials for producing electro-optic devices with nanometer-scale features that can be scaled for low-cost manufacturing. Many of our techniques are geared for compatibility with flexible plastic materials, leading to applications such as low cost disposable sensors, wearable sensors, flexible electronics, and flexible displays. Because our structures manipulate light at a scale that is smaller than an optical wavelength, we rely on computer simulation tools such as Rigorous Coupled Wave Analysis (RCWA) and Finite Difference Time Doman (FDTD) to model, design, and understand optical phenomena within photonic crystals and related devices.

In addition to fabricating devices, our group is also focused on the design, prototyping, and testing of biosensor instrumentation for high sensitivity, portability, and resolution. Advanced instruments enable high resolution imaging of biochemical and cellular interactions with the ability to monitor images of biochemical interactions as a function of time. Using the sensors and instrumentation, we are exploring new applications for optical biosensor technology including protein microarrays, biosensor/mass spectrometry systems, and microfluidics-based assays using nanoliter quantities of reagents. The methods and systems developed in the laboratory are applied in the fields of life science research, drug discovery, diagnostic testing, and environmental monitoring. -From Professor Cunningham's Faculty Profile

Cite this work

Researchers should cite this work as follows:

  • Brian Cunningham (2013), "[Illinois] ECE 416 DNA Microarrays I," https://nanohub.org/resources/17701.

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Time

Location

University of Illinois, Urbana-Champaign, IL

Submitter

NanoBio Node, Obaid Sarvana, George Michael Daley

University of Illinois at Urbana-Champaign

[Illinois] ECE 416 Lecture 31: DNA Microarrays
  • DNA Microarrays Lecture 22 How Gene Chips Work 1. DNA Microarrays Lecture 22 How… 0
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  • Problem •  How to analyze the expression of thousands of genes and to determine what possible physiological processes a gene may be related to 2. Problem •  How to analyze … 461.73763072326216
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  • DNA Review 3. DNA Review 464.62946036210229
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  • Central Dogma of Molecular Biology 4. Central Dogma of Molecular Bio… 587.97692916153983
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  • •  All your cells have the same genome, but each cell can express a different set of proteins •  Expression of proteins is variable, even for an individual cell –  Cell environment –  Stage in its life cycle –  Diseased versus healthy •  THE MILLION DOLLAR QUESTION: –  WHAT mRNA SEQUENCES ARE PRESENT IN A CELL? –  This tell us what genes are 5. •  All your cells have the… 766.36588152575143
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  • Solution: DNA Microarray •  Thousands of individual gene sequences applied in a grid of 6. Solution: DNA Microarray •… 795.00232905607311
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  • In this lecture •  How are DNA microarray spots made? •  How is detection performed? •  How is a DNA microarray chip used to measure gene expression? 7. In this lecture •  How are… 890.82905607312352
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  • DNA Detection Laser 8. DNA Detection Laser 907.19274037616447
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  • Method 1: Long Strand DNA Microarray •  1 ~1kb portion of the coding region of each gene to be analyzed is individually amplified by PCR •  A 9. Method 1: Long Strand DNA Micr… 1006.1186500263667
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  • DNA Array Spot-Making Methods 10. DNA Array Spot-Making Methods 1237.5656090701352
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  • Microarray Spotter 11. Microarray Spotter 1331.1609245913164
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  • Spots •  Spot application methods result in diameter ~100-200 µm •  Typical arrays are ~2x2 cm2 and have 6,000-10,000 spots •  Very difficult to always make perfect spots –  Repeatability from spot-to-spot –  Uniformity within a spot –  SEVERAL replicate spots are used within an array for each gene so an experiment is not ruined by a 12. Spots •  Spot application … 1492.0704869045526
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  • Method 2: Short Strand DNA Microarray •  DNA oligonucleotides of ~20bp in length are synthesized on the chip surface, one base pair at a time •  13. Method 2: Short Strand DNA Mic… 1577.4839602742134
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  • Affymetrix DNA Microarray 14. Affymetrix DNA Microarray 1664.6329759184389
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  • Affymetrix Microarray 15. Affymetrix Microarray 2136.0806380734753
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  • Light-Directed Spot Synthesis •  EXPENSIVE –  A unique photolithography 16. Light-Directed Spot Synthesis … 2215.7916153981369
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  • Detection •  The array of immobilized single-strand DNA 17. Detection •  The array of … 2220.3784056952013
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  • Tags •  Two fluorescent tags are commonly used –  Green •  CY3, λex= 532 nm –  Red •  CY5, λex= 635 nm •  Tags are attached to the detected (analyte) DNA 18. Tags •  Two fluorescent ta… 2279.6347776410616
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  • DNA Microarray 19. DNA Microarray 2368.1474336438741
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