Lesson Title: Teaching Microarray Analysis
Author: Rosanna Thomas

Rosanna Thomas, one of our outstanding HHMI teachers, has developed a lesson on Microarray Analysis that she used to train other teachers. The lesson includes PowerPoint slides that can be used by importing them into you files and showing them on the projected computer screen. (Download PowerPoint Presentation) The background for the teacher and student can be found on the website of the Office of Science Education at NIH. (view site) You can click on the section on the left hand column called "Print this issue." This is a PDF designed for direct download of a printable version of the stories and lesson. There also is a quick overview and a teachers guide to accompany the stories. (Download Teachers Guide 373K, PDF format)
Rosanna used this scientific background for her lesson along with other sources.

Microarrays are commonly known as DNA chips. A sample of an individual's DNA can be applied to the chip for testing. These devices allow investigators to analyize an individual's genetic sequence by comparing it to a normal genetic sequence that is also present on the surface of the chip. The non-matching genetic sequences show up by color mismatches under a viewing light. These non-matching sequences are indicators of mutations. This information would give the scientist the location of the flaws and possibly the clues to future development of an illness or abnormality.

The chip works on the concept of complementation of base pairs. Each of the strands in a bit of double-stranded DNA is complementary to the other. Adenine (A) is opposite thymine (T), cytosine (C) is opposite guanine (G). So, the sequence CCATGA would be complementary to GGTACT. Complementary DNA strands separate on gentle heating. They bind again when cooled. A DNA chip is made of many different DNA sequences stuck to a flat surface. Each spot on the surface contains a different sequence. You can use a single strand of DNA to "probe" a solution for that strand's complement: Put in the probe, slosh it around, and pull it out. If the complement is in there, it will bind onto the probe.

An expression array is made up of many different long DNA sequences, each complementary to every mRNA sequence that a certain cell can make. Researchers use these to study moment-to-moment changes in which genes are turned on or off. A researcher breaks a cell preparation open, extracts all the mRNA sequences it contains at that moment, and puts those on the expression array to see which ones are there. This tells the researcher which genes in the cell were turned on or being expressed, making mRNA-at the moment the cell broke open.

There are many future applications of this instrument. Soon it may be applied for a practical use in a doctor's office to guide them in appropriate preventive treatment for their patients. Doctors may be able to predict how their patient will respond to drugs that may be prescribed. The DNA chip may also be used by food safely checks for contamination. It could detect such things as Salmonella and other food-born pathogens. It also may be use by the law enforcement agencies to identify an individual and place them at the scene of the crime.

The lesson below is a simulation of a method being developed at a research lab at NIH. The Salmonella organism is currently resistant to approximately six different antibiotics. The detection of a strain of salmonella that causes problems such as diarrhea in humans will be a valuable tool in the food industry. The process can be used to show students how the DNA chip can be used for a very practical purpose. You may use these research objectives or any others that fit the use of the technology.

Research objectives:

• To identify specific virulence genes of Salmonella typhimurium DT 104 strain.
  
  
• To identify the DNA promoter regions that regulate expression of virulence genes with in infected human intestinal epithelial cells.
  
  
• To identify these genes by the use of DNA microarray technology.

Procedure:

1. Create or order DNA chip with copies of short single-stranded DNA molecules. ( The simulation of the slide is a series of colored papers lined up on the black board to be used for matching with the mRNA sequences found on cards to be given to the students)
   
   
2. Isolate mRNA from cells or tissue sample #1 ( give students cards with numbered genes such as, 050, 052, 053. Put the mRNA sequence on the reverse side for complementary matching to the single-stranded DNA on the blackboard.) See. Sample cards below.
   
   
3. Use a reverse transcriptase enzyme and fluorescent labeled nucleotides to make cDNA from the mRNA. (Using colored pieces of paper, make the complementary DNA sequence to match your card of mRNA. The teacher will list the base key on the board or on a handout) See examples below. #2
   
   
4. Hybridization: apply the cDNA mixture to your DNA chip. ( see the cDNA color key below) sample #3
   
   
5. Rinse off excess cDNA and scan for fluorescence.
   
   
6. Each fluorescent spot will indicate that the cDNA strand was complimentary to the strand on the DNA chip.
   
   
7. Tell which gene is expressed in tissue sample. (You will need to match the colored pieces of paper that represent the cDNA and then see which gene of mRNA it matches. If the mRNA is made then that is an indication of the expression of the gene.)

Sample #1 of mRNA sequences on cards

	050 -	AUG	UUU	AUG	GUA
	050 -	AUG	GAU	CCU	CAU
	050 -	AUG	CUC	AGA	UUA
	052 -	AUG	GAC	AGA	GGC
	052 -	AUG	GCG	CAA	AAA
	052 -	AUG	GCA	CCU	UUG
	053 -	AUG	CCC	AUU	CCG
	053 -	AYG	CCG	UUU	AAA

Sample #2

Adenine (A) is opposite thymine (T) and cytosine (C) is opposite guanine (G).
The sequence CCATGA would be complementary to GGTACT in the DNA.

Sample #3

Complementary Base pairs:

Red= Adenine (A)
Blue = thymine (T)
Yellow = Cytosine (C)
Green= guanine (G)

• View Student Worksheet

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