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DNA Chip for Genetic Analysis. Outline DNA and Genetic Analysis – Basic – Aim of detecting a target sequence – How to detect target sequences by affinity.

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Presentation on theme: "DNA Chip for Genetic Analysis. Outline DNA and Genetic Analysis – Basic – Aim of detecting a target sequence – How to detect target sequences by affinity."— Presentation transcript:

1 DNA Chip for Genetic Analysis

2 Outline DNA and Genetic Analysis – Basic – Aim of detecting a target sequence – How to detect target sequences by affinity reaction State-of-the-art – On surface detection on micrometer sites. Parallel reactions – Commercially available DNA chip: fluorescence Development –Label-free sensors –Label-free Fully-Electrical detection sensors

3 DNA and the Genome The Genome is the complete set of instruction to create and maintain an organism alive. DNA molecules (Deoxy-ribo- Nucleic-Acids) are support for the Genome. Every cell of our body (over a trillion) has the same strand of DNA (two meters long!!)

4 The molecular structure of DNA In 1953 J. Watson and S. Crick determined the double helix structure of DNA, owing to the important Xray diffraction works made by R. Franklin In 1962 Watson and Crick and Wilkins were awarded the Nobel Price for this discovery

5 Base pairing Specific recognition of DNA (RNA) is based on base pairing “the specific base pairing immediately suggests a possible copying mechanism for the genetic material” (Watson and Crick, 1953) Sequence Number of Complementary bases Percentage of G-C couples Solution Ionic Force Temperature The Affinity Reaction depends on:

6 Lab-on-Chip  Miniaturized integrated components  Advantages:  Less reagents  Time saving  Increase throughput

7 Point-of-care Full system from blood plasma sample or saliva to diagnostic information Development of building blocks of biological sample treatment True lab-on-chip system

8 Large scale revolution 1900-industrial revolution  2000-biological revolution

9 Aim of detecting a target sequence To Determine a part of the DNA sequence (eg: Genome sequencing) SEQUENCING DNA analysis + To understand the molecular bases of the (human) phenotype EXPRESSION PROFILING mRNA or protein analysis GENOTYPING Statistic of the presence of single base mutations in a population To have information on the health of living being DIAGNOSTICS &

10 Localized Recognition Sample to test (TARGET) Oligonucleotide of known sequence (PROBE) Substrate Syntetic Chemistry Surface physical-chemistry Microfabrication technology Know-how Implementation of a sensing method or a transduction system

11 B.P. for Target Recognition on Surfaces Surface Functionalization Cross-linker

12 Foundamental Innovation in Genetic Analysis: DNA CHIPS Possibility of attach, localize and/or address receptors onto a substrate in a very precise and dense way Multi-site detection (more fast and simultaneous detection) Miniaturized devices (less sample quantity and reagent cost, mass production) Micro-arrays Microfabricated two-dimentional structures for parallel analysis

13 DNA chips The term DNA chip or DNA microarray refers to the systematic arrangement of biomolecular probes such as DNA molecules on a solid surface (e.g. glass, metal, or silicon wafer).

14 Sequence for Genotyping-Diagnostics Yellow dyes correspond to probes that have hybridized with green- and red labeled targets.

15 Slide scanning Fluorochrome excitation at a selected wavelength GenePix Axon Instrument (http://www.axon.com/GN_Genomics.html)

16 How does a microarray scanner work ?

17

18 Photocatode and photomultiplier

19 Image acquisition Cy5 wavelength Cy3 wavelength

20 Micro-Technology for immobilization of receptors Mechanical Micro-spotting Bubble Jet Technology Inkjet technology Electric field-assisted Photolitography

21 Mechanical micro-spotting Up 40,000 different probe on a microscope slide (3000 spot/cm 2 ) Stanford University (California) pinhead (~ micrometers)

22 Bubble Jet Technology The Bubble Jet method produces a consistent number of uniform spots and yields a spot density of up to 20000/cm 2. Its chemical adhesion also eliminates the problem of peeling. Canon

23 InkJet Printing Deliver small amounts of liquids with good spatial accuracy up to 14,000 cDNAs/array. IBM and Agilent Technologies

24 Electric-field assisted Nanogen, NanoChip® San Diego Linker Molecules Metal electrode + Serial Approach

25 Photolitography Higher Density features/cm 2 Each feature hosts millions of identical DNA molecules Number of masks 25*4=100 Affymetrix, GeneChip® Santa Clara (California)

26 Recent developments Drawbacks of state-of-the-art methods Indirect (Labelled) methods Detection of a non electrical parameter Label-free Techniques Label-free and Fully-electrical Techniques Fully-electrical detection Techniques

27 Label-free sensor Surface Stress Mass Optical Properties Electrical Properties Variation of:

28 Label-free sensor (surface stress) Biochemically induced surface stress Cantilever arrays Gold and thiolated oligonucleotide probes

29 Label-free sensor (surface stress)  eight identical silicon cantilevers  250 µm pitch  length of 500 µm  width of 100 µm  thickness of 0.5–1 µm

30 Label-free sensor (mass) The quartz crystal microbalance (QCM) is an extremely sensitive mass sensor, capable of measuring mass changes in the nanogram range. QCMs are piezoelectric devices fabricated on a thin plate of quartz, with gold electrodes fixed to each site of the plate

31 Label-free Optical Molecular Layers on Porous Materials Porous Silicon or Aluminium: Very High Surface/Volume ratio

32 Label-free Optical Interferometry The silicon oxide surface of the porous layer can be modified to host molecular recognition elements Reflection of white light ( W- lamp source) at the top and bottom of the PSi layer results in an interference pattern (Fabry-Perot fringes). The reflectometric interference spectrum is sensitive to the refractive index of the PSi matrix. Interactions of the molecular species with their recognition partners on the surface induce a change in the refractive index

33 Recent developments Drawbacks of state-of-the-art methods Indirect (Labelled) methods Detection of a non electrical parameter Label-free Techniques Label-free and Fully-electrical Techniques Fully-electrical detection Techniques

34 Label-free/Fully-Electrical Techniques Chemical Interactive Material Electrolyte


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