DNA Microarray:

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DNA Microarray

• A Recombinant DNA Method

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Basic Steps to Microarray

• Obtain cells with genes that are needed for
  analysis.
• Isolate the mRNA using extraction buffer.
• Remove the buffer with RNA.
• The mRNA will be 5-3 with repeating AAA..end.
• Convert each mRNA into colored cDNA.
• Targets labeled with fluorophores.

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Basic Steps to Microarray

• With mRNA degraded reverse cDNA created.
• Mix colored samples of cDNA.

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Microarray or DNA Chip

• Every spot on the chip represents a different
  coding sequence from different genes.
• Each spot on the chip is made of a DNA probe that
  can pair with the cDNA that was created.

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Basic Steps to Microarray

• Incubation with the mixed cDNA and the chip DNA
  will yield some pairing.
• Wash off unbound cDNA to see what has bound to
  the microarray.

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Visualizing Bound cDNA

• 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.
• The information and images are then sent to the
  computer for analysis.

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Analyzing the Data

• Creates a ratio image.
• Green images signal expression in one condition.
• Red images signal expression in one condition.
• Yellow images signal expression in both
  conditions.

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Capacities

• Not limited to human genetic material.
• Can be used for all species.
• Can display thousands of different genes.
• Pending chip size.
• 100s nucleotides (lt1 cm2)
• 100,000s nucleotides (about 10 cm2)
• Allows the study of multiple genes at once.

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Types of Microarray

• Pre-synthesized nucleic acids.
• Bought cDNA.
• Oligonucleotide synthesis in situ.
• Creating cDNA in question.

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Microarray Application

• Identifying genes and gene mutations of
  different types of diseases such as cancer.
• Identifying the expression level, or quantity of
  genes (mutated or not).

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Definitions

• Target - the nucleic acid (cDNA) sample whos
  identity and quantity are being measured.
• Fluorophore usually green and red labels
  attached to the target to enable visualizing
  expression.
• Microarray works as reverse hybridization method
  converting from mRNA to cDNA 3-5 with TTTTend.
• Probe an attached nucleic acid with a known
  sequence (the DNA chip).

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Resources

• Strachan, Tom Read, Andrew. Human Molecular
  Genetics 3. pp 175-178. New York. Garland
  Publishing. 2004.
• Campbell,A. Malcom. Microarray Animation.
  Davidson College. 2001. www.bio.davidson.edu/cours
  es/genomics/chip/chip.html
• http//cmgm.stanford.edu
• http//www.gene-chips.com/