DNA Microarray: A Recombinant DNA Method. Basic Steps to Microarray: Obtain cells with genes that are needed for analysis. Isolate the mRNA using extraction.

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Presentation transcript:

DNA Microarray: A Recombinant DNA Method

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. Targetsfluorophores

Basic Steps to Microarray: With mRNA degraded reverse cDNA created.reverse Mix colored samples of cDNA.

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. probe

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.

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.

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.

Capacities: Not limited to human genetic material. Can be used for all species. Can display thousands of different genes. Pending chip size. 100’s nucleotides (<1 cm^2) 100,000’s nucleotides (about 10 cm^2) Allows the study of multiple genes at once.

Types of Microarray: Pre-synthesized nucleic acids. Bought cDNA. Oligonucleotide synthesis in situ. Creating cDNA in question.

Microarray Application: Identifying gene’s and gene mutations of different types of diseases such as cancer. Identifying the expression level, or quantity of genes (mutated or not).

Definitions: Target - the nucleic acid (cDNA) sample who’s identity and quantity are being measured. Target Fluorophore – usually green and red labels attached to the target to enable visualizing expression. Fluorophore Microarray works as reverse hybridization method converting from mRNA to cDNA 3’-5’ with TTTT…end. Microarray Probe – an attached nucleic acid with a known sequence (the DNA chip). Probe

Resources: Strachan, Tom; Read, Andrew. Human Molecular Genetics 3. pp New York. Garland Publishing Campbell,A. Malcom. Microarray Animation. Davidson College