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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). 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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slide1
Example of a DNA array used to studygene expression (note green, yellow red colors; also note that only part of the totalarray is depicted)

http://www.biomed.miami.edu/arrays/images/agilent_array.jpg

slide2
DNA Arrays:

Two Purposes

Genotype individuals on polymorphisms(read text for example).

Study gene expression (these slides).

slide3
Methods:Studying Gene Expression

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.

slide4
cDNAgene 1

cDNAgene 2

cDNAgene 3

cDNAgene 4

cDNAgene 5

DNA Array: Single-stranded copy DNA foreach gene in a different well.

slide5
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.

slide6
Extract mRNA from Control Cells

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)

slide7
Methods:Studying Gene Expression

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.

slide8
Control cDNA

Experimental cDNA

slide9
Methods:Studying Gene Expression

Step 5: Extract a smallamount in a pipette.

slide10
Methods:

Step 6: Insert into first well.

slide11
Methods:

Step 7: Extractmore cDNA and …

… insert intosecond well, etc.

slide12
Methods:

Step 8ff: Continue until all wells arefilled.

slide13
Methods:

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

slide14
Result:

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)

slide15
Methods:

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

slide16
Only in

controlcells

Mostly in

controlcells

Mostly in

experimentalcells

Only in

experimentalcells

Same inboth cells

Results:

The colors denote the degree of expression in the experimental versus the control cells.

Gene not expressed in control or in experimental cells

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