Example of a DNA array used to study gene expression (note green, yellow red colors; also note.

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Example of a DNA array used to study gene expression (note green, yellow red colors; also note that only part of the total array is depicted)

DNA Arrays: Two Purposes 1. Genotype individuals on polymorphisms (read text for example). 2. Study gene expression (these slides).

Step 1: Create a DNA array (gene “chip”) by placing single-stranded DNA for each gene to be assayed into a separate “well” on the chip. Methods: Studying Gene Expression

cDNA gene 1 cDNA gene 2 cDNA gene 3 cDNA gene 4 cDNA gene 5 DNA Array: Single-stranded copy DNA for each gene in a different well.

Methods: Studying Gene Expression Step 2: Extract mRNA from biological tissues subjected to an experimental treatment and from the same tissue subjected to a control treatment. Step 3: Make single-stranded DNA from the mRNA using “color coded” nucleotides.

Extract mRNA from Control CellsExtract mRNA from Experimental Cells Make single-stranded cDNA using green nucleotides Make single-stranded cDNA using red nucleotides cDNA = complementary DNA (DNA synthesized from RNA)

Step 4: After making many DNA copies of the RNA, extract an equal amount of cDNA from the controls & experimentals and place it into a separate container. Methods: Studying Gene Expression

Control cDNAExperimental cDNA

Step 5: Extract a small amount in a pipette. Methods: Studying Gene Expression

Step 6: Insert into first well. Methods :

… insert into second well, etc. Methods: Step 7: Extract more cDNA and …

Methods: Step 8ff: Continue until all wells are filled.

Methods: Step 9: Allow to hybridize, then wash away all single-stranded DNA.

Result: (1) Some wells have no color-coded cDNA (no mRNA in either type of cell) (2) Some wells have only red (i.e., expressed only in experimental cells) (3) Some wells have only green (i.e., expressed only in control cells) (4) Some wells have both red and green in various mixtures (expressed in both experimental and control cells)

Methods: Step 10: Scan with a laser set to detect the color & process results on computer.

Results: The colors denote the degree of expression in the experimental versus the control cells. Gene not expressed in control or in experimental cells Only in control cells Mostly in control cells Only in experimental cells Mostly in experimental cells Same in both cells