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). http://www.biomed.miami.edu/arrays/images/agilent\_array.jpg. DNA Arrays: Two Purposes. Genotype individuals on polymorphisms (read text for example).
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Genotype individuals on polymorphisms(read text for example).
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.
DNA Array: Single-stranded copy DNA foreach gene in a different well.
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 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.
Step 5: Extract a smallamount in a pipette.
Step 6: Insert into first well.
Step 7: Extractmore cDNA and …
… insert intosecond well, etc.
Step 8ff: Continue until all wells arefilled.
Step 9: Allow to hybridize, then wash away all single-stranded DNA.
Some wells have no color-coded cDNA (no mRNA in either type of cell)
Some wells have only red (i.e., expressed only in experimental cells)
Some wells have only green (i.e., expressed only in control cells)
Some wells have both red and green in various mixtures (expressedin both experimental and control cells)
Step 10: Scan with a laser set to detect the color & process results on computer.
Same inboth cells
The colors denote the degree of expression in the experimental versus the control cells.
Gene not expressed in control or in experimental cells