fishing for genes_dna microarrays in the classroom

www.scienceinschool.org44 Science in School Issue 12 : Summer 2009

Fishing for genes: DNA microarrays in the classroom

Anastasios Koutsos, Alexandra Manaia, andJulia Willingale-Theune bring a sophisticatedmolecular biology technique into the classroom

Fishing for genes one step at a time: a short story

Once upon a time in a small village, there was a man. Every day he went to a pond to

catch fish for his dinner. One day it would be a catfish, the next day an eel — sometimes it

would be a different fish each day of the week. Then one day he wondered how many differ-

ent species of fish there were in the pond, and how many of each species. How could he

find out? It was obvious that catching one fish at a time wouldn’t work — for all he knew the

pond might contain thousands of fish. So he browsed the Internet for ideas and found a

book listing 20 000 species of freshwater fish, with the bait used to catch them.

Eventually, he thought of a complex but clever solution: he cast 20 000 fishing lines, each

with an array of hooks, into the pond. Using the information from his reference book on fish,

he attached a particular type of bait to the hooks on each line to attract a particular species

of fish, and in this way created different lines to catch all fish species. The rest was easy: he

waited for the fish to find their bait and collected his catch.

[Note – this is a fictitious story: about 9000 species of freshwater fish are known today, and there is not a different bait for

each one.]Imag

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A scanned microarray

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Teaching activities

www.scienceinschool.org 45Science in School Issue 12 : Summer 2009

Monitoring gene expression: theanalytical power of DNAmicroarrays

Now that the draft sequence of thehuman genome has been published(along with the genome sequences ofmany other organisms), the challengeis to work out what the individualgenes do, and to do so, scientists aredesigning sophisticated tools. Onesuch tool is the DNA microarray.

Our story of the fisherman is ficti-tious, but the principles used by theman to find out how many fish therewere in the pond are very similar tothe principles underlying DNAmicroarray technology – a technologythat has revolutionised the way scien-tists look at living processes.

Until the 1990s, biologists couldmeasure the activity of only a handfulof genes in a cell at any given time. Agene is active when it is being tran-scribed to its messenger molecule,mRNA, which in turn is used to makeone or several proteins.

Frustrated that they could not lookat the expression of genes in concertin a single cell, scientists began tosearch for ways to simultaneouslymeasure the activity of all genes in acell. To do this, they needed to findout how many different mRNAs werepresent in a cell at any given time,and which genes those mRNAs corre-sponded to. It was this revolutionaryidea that culminated in the develop-ment of DNA microarrays. Those sci-entists created something similar tothe 20 000 fishing lines in our story.They took a piece of treated glass sim-ilar to a microscope slide and immo-bilised batches of single strands ofDNA on its surface, each batch corre-sponding to a single gene. TheseDNA strands could then act as bait toattract the corresponding mRNA mol-ecules belonging to the gene, just likethe specific bait attracted a particulartype of fish. When viewed with thenaked eye, those groups of DNAstrands look like small dots. Eachglass slide is printed with thousands

Gene name (circles) Gene colour

alexander fleming 3 0 3

barbara mcclintock 7 1 6

francis crick 18 9 9

jacques monod 4 4 0

james watson 0 0 0

john kendrew 3 2 1

leo szilard 12 9 3

maurice wilkins 6 3 3

rosalind franklin 2 1 1

thomas morgan 12 4 8

of dots arranged in an array, makingup a DNA microarray.

Microarray experiments can take a lot of time. Scientists need to pre-pare the slides in advance and thenanalyse the information from thou-sands of spots. The analysis requiresexpertise in mathematics andadvanced statistics, and can takemonths. To illustrate the underlyingconcepts of this technology in theclassroom, you need to go virtual.

The real and virtual microarrayexperiment step-by-step

The virtual microarrayw1, developedby the European Learning Laboratoryfor the Life Sciences (ELLS)w2 at theEuropean Molecular BiologyLaboratoryw3, is an educational activi-ty that uses everyday materials tosimulate a microarray experiment inthe classroom. The game simulatesthe different steps that researcherstake in performing microarray experi-ments and in analysing the results.The activity is aimed at 16- to 18-year-olds and can be used to complement

lessons on genetics, cell developmentand genetic diseases.

The following step-by-step guidedescribes how a real microarrayexperiment is performed, alongsidethe steps of the virtual microarray. Inthis activity, the virtual microarraywill be used to detect differences ingene expression between a normalcell and a cancerous cell. You candownload technical information andsuggestions on how to perform thevirtual microarray experiment in theclassroom from the ELLSTeachingBase (‘In the classroom’PDF)w1.

If prepared in advance, the basicexercise takes about one hour. Thediscussion exercises could be used inclass or set for homework.

Materials

· A mat large enough to accommo-date 10 circles (35 cm in diameter)that represent the DNA spots

· 5 red, 5 green and 5 yellow circles,35 cm in diameter, made ofcoloured plastic or paper.

Table 1: The names and colours of the genes and the amount of mRNA that is usedfor each gene

Total mRNAamount for thisgene (numberof Velcro strips)

Amount ofmRNA in normal cell (green torches)

Amount of mRNAin cancerous cell (red torches)



· A pen/marker to draw the circlesand label them

· Velcro in different colours (a colourfor each gene), which you canmake yourself by colouring whiteVelcro with felt-tip pens

· 33 green and 34 red torches, plus afew extra. We use small “promo-tional flashlights” that can be pur-chased relatively cheaply.However, students can also bringtheir own torches and cover themwith red or green paper. You mighthave to stand the torch in a smallmug or pencil holder to ensure thelight is emitted from the floor tothe ceiling. For variants usingsmaller numbers of torches, see theELLS TeachingBase (‘In the class-room’ PDF)w1.

· 2 boxes to hold about 35 torches each

· Printouts of Table 1 (available fordownloadw4)

Use a room or classroom that can bedarkened (with curtains or blinds).

ManufacturingReal

DNA microarrays are printed ontosmall glass slides, similar to micro-scope slides. Placing minute spots onthis glass surface by hand is tricky, soscientists have developed specialprinting robots. Their printer headshave special pins that carry an aque-ous solution of DNA from reservoirsand print it onto the glass surface in agrid of small spots. By controllingthose robots, you can control all the

Manufacturing

A microarray beingprinted

Close-up of amicroarrayprinter head

Microarrayslides

Images courtesy of EM

BL Photolab


Teaching activities


features of the array: the number, sizeof and distance between the spots. Asmany as 20 000 spots can be printedonto a slide, and each spot containsbillions of copies of DNA for a specif-ic genew4.

Virtual1. Draw 10 circles of about 35 cm in

diameter on the mat to representthe DNA spots.

2. Label each circle according to Table1 to represent a gene named after afamous scientist.

3. According to the second column inTable 1, stick at least the correspon-ding number of Velcro strips (thesoft, loop side only) of the samecolour in each circle (if there aremore strips than needed, it doesn’tmatter). The Velcro pieces corre-spond to the DNA strands andeach colour represents DNA fromone gene. Note down the colour inthe table.

mRNA extraction

Real In a microarray experiment, the first

step is to extract mRNA from cells ofthe two conditions under study, suchas normal and cancerous liver tissue.mRNA is extracted by a standard labprocedure and then labelled with afluorescent dye: for example, greenfor the normal cells, and red for thecancerous ones.

Virtual1. The fluorescently labelled mRNA

molecules are represented by smalltorches. Each torch corresponds toa single chain of mRNA from aparticular gene. Green torches rep-resent mRNA extracted from a nor-mal cell; red torches representmRNA extracted from a cancerouscell.

2. According to Table 1, stickcoloured Velcro (hook side) on theunderside of the correspondingnumber of green and red torches,using one colour of Velcro pergene.

3. Label a few extra torches eitherwith multicoloured pieces of Velcroor Velcro in a colour that is not rep-resented in the virtual array. Thesewill represent mRNA moleculesthat bind only weakly or not at allto the DNA on the mat, i.e. the 10genes.

4. Place all green torches in one box(normal cell) and all red ones inanother (cancerous cell).

Hybridisation

Real In the hybridisation step, control

(green) and test (red) mRNAs aremixed together, and the mRNA mole-cules are flooded over the surface ofthe microarray to bind to their com-plementary, immobilised DNAstrands. mRNA molecules with asequence that is not complementaryto any of the genes will not bind.Those with a sequence partially simi-lar to DNA strands from one genewill bind weakly (non-specific

hybridisation). These molecules willbe removed by a subsequent washingstep.

Virtual 1. Extract the torches (mRNA mole-

cules) from the boxes (cells), lookat the colour of the Velcro tape andmatch it to the colour of the Velcroon the array (the mat).

2. Some mRNA molecules havecolours that do not match any ofthe genes, and some have multi-coloured tape that matches weaklyto some genes.

3. Remove the unbound and weaklybound torches from the array byperforming a virtual washing step.

Scanning

Real Now it is time to look at the results

of the experiment to see whichmRNA has hybridised with whichgene. Because mRNA molecules arenot visible to the naked eye, scientistscan only estimate the amount ofhybridised mRNA indirectly, bymeasuring the fluorescence of each

Hybridisation Scanning

Images courtesy of EMBL Photolab

Image courtesy of EM

BL Photolab

A scanned microarray (detail)


spot. Using a special laser scanner,red and green fluorescence is detectedindependently and two images areproduced. Putting these two imagestogether results in a characteristic pic-ture with coloured spots (see imageon page 47). A red spot indicates thatthe gene is active in the cancerouscell, and a green spot indicates thatthe gene is active in the normal cell. Ayellow spot indicates that the gene isequally active in both cells types.

Virtual 1. Switch on the torches and darken

the room.

2. Ask the students to describe whatthey see for each gene. How manygreen and red torches does eachgene have? In other words, is themRNA expression different in thenormal and cancerous cell for dif-ferent genes (i.e. if there are thesame number of red and greentorches in each circle, the ex pre ssionis un changed)? Encourage the stu-dents to draw their own conclusions.

3. Explain to the students that thered, green and yellow signals of thereal microarray depend on theamount of mRNA from those twocells. Note: for light, red and green

mixed together give yellow. Forpigments, red and green producepurple.

4. Switch off the torches, hand out thecoloured circles, and ask the stu-dents to indicate which spotswould show up as red, green oryellow in a real microarray,depending on the number of torches.

Analysis

Real Microarray analysis starts with

measuring the fluorescent intensity ofeach spot. This is done automaticallyby a scanner. Although analysis of theresulting images depends on the spe-cific experiment, a typical step is togroup genes with similar behaviour,in a process called clustering. Afterclustering, the scientists attempt todefine similarities and differencesamong the genes belonging to thesame clusters.

Virtual1. Using Table 1, ask the students to

cluster the genes into groupsaccording to similar behaviour inthe microarray. (See ‘Teacher’sguide to clustering exercises’ and‘Clustering exercises for the class-room’ in the ELLS TeachingBASEw1).

2. Discuss the different solutions ofclustering genes. Mention to thestudents that there is no wrong orright clustering solution, but thatthe conclusions drawn from eachcluster can be different.

3. Ask the students to search forinformation about the scientistsafter whom the genes were named,to find out if the genes in a clusterhave something in common.

4. Depending on the clustering, you may come up with a group of the scientists who discovered the double helix, a cluster ofgeneticists, a cluster of scientistswho did research on micro-organisms, or even scientistsresponsible for the establishment of


Image courtesy of EMBL Photolab


the European Molecular BiologyLaboratory.

Conclusion

After performing the virtualmicroarray experiment, students candiscuss how microarray experimentsare used in biomedical research, andthe medical and ethical implicationsof this technology. For more informa-tion, there is a ‘Reading club’ sectionin the ELLS TeachingBASEw1, withsimplified versions of originalresearch articles describing howmicroarrays were used to answerimportant biological questions.

AcknowledgementsThe authors would like to thank

Rosanna de Lorenzi for playing andrefining the activity. They would alsolike to express their obligation toMehrnoosh Rayner for commentingon the article, Thomas Sandmann forhelp in the initial stages of the virtualmicroarray, and John Watson for help-ful discussions.

Web referencesw1 – For more information on the vir-

tual microarray activity in English,German and Greek, see theTeachingBASE on the ELLS website– a freely available collection ofmolecular biology teachingmodules designed for teachers and students: www.embl.org/ells

The following PDF materials in thevirtual microarray section are par-ticularly relevant to this article:

In the classroom

Reading club

Teacher’s guide to clustering exercises

Clustering exercises for the classroom

w2 – For more information about theEuropean Learning Laboratory forthe Life Sciences (ELLS), see:www.embl.org/ells

w3 – For more information about theEuropean Molecular BiologyLaboratory, see: www.embl.org

w4 – A video of a robot printingmicroarrays at the EuropeanMolecular Biology Laboratory and aWord document of Table 1 can be downloaded from the Science inSchool website:www.scienceinschool.org/2009/issue12/microarray

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fishing for genes_dna microarrays in the classroom

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