DNA Microarray:

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DNA Microarray:

  1. 1. DNA Microarray: A Recombinant DNA Method
  2. 3. Basic Steps to Microarray: <ul><li>Obtain cells with genes that are needed for analysis. </li></ul><ul><li>Isolate the mRNA using extraction buffer. </li></ul><ul><ul><li>Remove the buffer with RNA. </li></ul></ul><ul><ul><li>The mRNA will be 5’-3’ with repeating AAA..end. </li></ul></ul><ul><li>Convert each mRNA into colored cDNA. </li></ul><ul><ul><li>Targets labeled with fluorophores . </li></ul></ul>
  3. 4. Basic Steps to Microarray: <ul><li>With mRNA degraded reverse cDNA created. </li></ul><ul><li>Mix colored samples of cDNA. </li></ul>
  4. 5. Microarray or DNA Chip: <ul><li>Every spot on the chip represents a different coding sequence from different genes. </li></ul><ul><li>Each spot on the chip is made of a DNA probe that can pair with the cDNA that was created. </li></ul>
  5. 6. Basic Steps to Microarray: <ul><li>Incubation with the mixed cDNA and the chip DNA will yield some pairing. </li></ul><ul><li>Wash off unbound cDNA to see what has bound to the microarray. </li></ul>
  6. 7. Visualizing Bound cDNA: <ul><li>The slide with the microarray chip is placed inside a dark box where it is scanned with a high resolution laser that detects the bound fluorescent labels. </li></ul><ul><ul><li>The information and images are then sent to the computer for analysis. </li></ul></ul>
  7. 8. Analyzing the Data: <ul><li>Creates a ratio image. </li></ul><ul><li>Green images signal expression in one condition. </li></ul><ul><li>Red images signal expression in one condition. </li></ul><ul><li>Yellow images signal expression in both conditions. </li></ul>
  8. 9. Capacities: <ul><li>Not limited to human genetic material. </li></ul><ul><ul><li>Can be used for all species. </li></ul></ul><ul><li>Can display thousands of different genes. </li></ul><ul><ul><li>Pending chip size. </li></ul></ul><ul><ul><ul><li>100’s nucleotides (<1 cm^2) </li></ul></ul></ul><ul><ul><ul><li>100,000’s nucleotides (about 10 cm^2) </li></ul></ul></ul><ul><li>Allows the study of multiple genes at once. </li></ul>
  9. 10. Types of Microarray: <ul><li>Pre-synthesized nucleic acids. </li></ul><ul><ul><li>Bought cDNA. </li></ul></ul><ul><li>Oligonucleotide synthesis in situ . </li></ul><ul><ul><li>Creating cDNA in question. </li></ul></ul>
  10. 11. Microarray Application: <ul><li>Identifying gene’s and gene mutations of different types of diseases such as cancer. </li></ul><ul><li>Identifying the expression level, or quantity of genes (mutated or not). </li></ul>
  11. 12. Definitions: <ul><li>Target - the nucleic acid (cDNA) sample who’s identity and quantity are being measured. </li></ul><ul><li>Fluorophore – usually green and red labels attached to the target to enable visualizing expression. </li></ul><ul><li>Microarray works as reverse hybridization method converting from mRNA to cDNA 3’-5’ with TTTT…end. </li></ul><ul><li>Probe – an attached nucleic acid with a known sequence (the DNA chip). </li></ul>
  12. 13. Resources: <ul><li>Strachan, Tom; Read, Andrew. Human Molecular Genetics 3 . pp 175-178. New York. Garland Publishing. 2004. </li></ul><ul><li>Campbell,A. Malcom. Microarray Animation. Davidson College. 2001. www.bio.davidson.edu/courses/genomics/chip/chip.html </li></ul><ul><li>http://cmgm.stanford.edu </li></ul><ul><li>http://www.gene-chips.com/ </li></ul>

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