DNA Microarray - Southwest Center for Microsystems Education

DNA Microarray Knowledge Probe (Pre-test) DNA Microarray PK Activities (4) Participant Guide

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DNA Microarray Learning Module This Learning Module contains the following SCOs (Sharable Content Objects): Knowledge Probe (KP or Pre-assessment) Primary Knowledge (Reading material) DNA Hybridization Activity DNA Microarray Terminology Activity DNA Microarray Model Activity DNA Microarray – An Ethical Dilemma? Activity Final Assessment Target audiences: High School, Community College, University

Support for this work was provided by the National Science Foundation's Advanced Technological Education (ATE) Program through Grant #DUE 0902411. Any opinions, findings and conclusions or recommendations expressed in this material are those of the authors and creators, and do not necessarily reflect the views of the National Science Foundation. Copyright © by the Southwest Center for Microsystems Education and The Regents of the University of New Mexico Southwest Center for Microsystems Education (SCME) 800 Bradbury Drive SE, Suite 235 Albuquerque, NM 87106-4346 Phone: 505-272-7150 Website: www.scme-nm.org

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Page 2 of 5 DNA Microarray Knowledge Probe

DNA Microarrays Knowledge Probe Participant Guide Introduction The purpose of this knowledge probe is to determine your current understanding of the applications, operations, interpretation, and fabrication of DNA microarrays. It is not a test. Answer each question to the best of your current knowledge. 1. Cytosine, Guanine, Adenine, and Thymine are the _______________ of a DNA molecule. a. Oligonucleotides b. Nucleotides c. Nitrogenous bases d. Genomes 2. A cytosine, guanine, adenine or thymine with a sugar and a phosphate is called ______________. a. Oligonucleotide b. Nucleotide c. Polymorphism d. Genome 3. Which of the following is NOT a valid base pair sequence? a. A-T, T-A, C-G, A-T b. T-A, C-G, C-T, T-A c. T-A, T-A, G-C, A-T d. C-G, G-C, C-G, A-T 4. A DNA microarray uses synthetic ______________________ as probes to capture target molecules from test and control samples. a. Oligonucleotides b. Nitrogenous bases c. Polymorphisms d. Genomes 5. DNA microarrays depend on which of the following processes to occur on the surface of the microarray in order to accurately analyze the DNA of the control and test samples? a. Replication b. Transcription c. Reverse Transcription d. Hybridization



6. Which of the following applications do NOT use DNA microarrays? a. Genome studies of various species b. Identification of specific genes for specific diseases c. Study how genes react to specific drugs or drug dosages d. Identification of specific viruses or bacteria e. All of the above applications use DNA microarrays 7. Which of the following BEST explains the process that takes place on the surface of a DNA microarray? a. Hybridization occurs between a synthetic oligo probe on the array and a complementary ssDNA from the control or test sample b. Hybridization occurs between a ssDNA from the control sample and a complementary DNA from the test sample c. DNA transcription divides a DNA molecule from the test sample into a ssDNA and RNA d. A copy DNA is made from the test sample’s RNA using reverse transcription 8. Which of the following BEST describes a GeneChip®? A grid or array consisting of thousands or millions of … a. genes from a specific organism strategically placed on a glass or silicon substrate using an inkjet printing process b. synthetic oligos that were fabricated using an inkjet printing process c. synthetic oligos that were fabricated using a photolithography process d. ssDNA from a control and a test sample strategically placed on a silicon substrate using a photolithography process 9. The photolithography fabrication process of a DNA microarray using masks requires which of the following components? a. Glass substrate, a set of masks, UV light, hundreds of oligonucleotide solutions b. Silicon substrate, a set of masks, UV light, four nucleotide base solutions with blocking agent c. Silicon substrate, a set of masks, UV light, hundreds of oligonucleotide solutions with blocking agent d. Glass substrate, a set of masks, UV light, a blocking agent, four oligo solutions 10. Which of the following BEST describes the process steps of the photolithography process used for DNA microarray fabrication? a. Coat, align, expose, develop b. Protect, deprotect, develop c. Protect, deprotect, addition d. Coat, deprotect, addition



11. In the photolithography process of a DNA microarray using masks, each mask identifies the placement location of which of the following components? a. A specific oligonucleotide b. A specific DNA sequence c. A specific DNA hybrid d. A specific nucleotide base 12. What is the purpose of the UV light in the photolithography process of a DNA microarray? a. To remove the blocking agent from the top of the oligo chain b. To add the blocking agent to the top of the oligo chain c. To add a nucleotide base to the top of the oligo chain d. To block a specific position from the placement of a nucleotide base 13. Which of the following prevents the addition of a nucleotide base to specific features during the addition step of photolithography fabrication? a. Mask b. Blocking agent c. UV light d. Fluorescent tag 14. In the interpretation of a DNA microarray, what color would indicate the presence of cDNA from the control sample as well as the test sample? a. Yellow b. Red c. Green d. Black 15. In the interpretation of a DNA microarray, what color would indicate the presence of cDNA from only the test sample? a. Yellow b. Red c. Green d. Black 16. DNA microarrays are fabricated with “positive and negative control features”, features that verify the validity of the test. Which of the following would indicate an “invalid” test? a. Positive control feature with both control and test sample genes b. Positive control feature with both control and test sample genes AND negative controls with no hybrids shown c. Negative control feature with genes from neither the control or test sample d. Negative control feature with a gene or genes from either the control or test sample

Support for this work was provided by the National Science Foundation's Advanced Technological Education (ATE) Program.



DNA Microarrays Primary Knowledge Unit Participant Guide Description and Estimated Time to Complete This DNA Microarray unit provides an overview of the DNA microarray and the GeneChip©, devices used to identify specific DNA (Deoxyribonucleic acid)sequences in a sample. A DNA Microarray consists of thousands of DNA or gene probes used to hybridize target molecules consisting of genomic DNA (gDNA) or cDNA (complementary or copy DNA, copied from messenger RNA). The detection of target DNA hybridized to a microarray is used in the medical or research lab to assess the types of mutations found in gDNA or the levels of gene expression, “information about all Two GeneChips© by Affymetrix with messenger RNAs that are projected results.Image courtesy of made in various cell Affymetrix. types”.(NCI, 2010) This unit explains what a DNA Microarray does, how it works, how it is used, and how it is fabricated. There are several names used for microarrays, depending on the method of fabrication and its specific application: GeneChips®, DNA chip, DNA microarray, genome chip, and biochip. In this unit we will stick to the term DNA microarray, however, when quoting from other sources, you might see GeneChip© or another term. To get the most out of this unit, you should have a basic understanding of DNA structure and function, DNA transcription and hybridization (or “base-pairing rules”), and terminology associated with genomes (an organism’s genetic material). Some knowledge of bioMEMS would also be beneficial. If you need to review, it is highly recommended that you complete the following SCME Learning Modules prior to this one.  BioMEMS Overview  BioMEMS Applications Overview  DNA Overview  DNA to Protein Overview These learning modules provide you with a review of DNA structure and function, DNA transcription and hybridization, as well as an overview of bioMEMS devices and their applications. Many of these terms may be new to you; therefore, a glossary is provided at the end of this unit for your reference. Estimated Time to Complete Allow approximately 30 minutes to an hour to read through this unit.

Introduction The Human Genome Project(1990 through 2003) introduced an era in which individualized approaches to medicine are possible through an analysis of a person’s DNA, or most specifically their gene expression levels and exact gene sequences (topics we’ll discuss in this unit). One outcome of this project was that the human genome (complete set of DNA) encodes approximately 30,000 genes. A person’s specific genes are stored in each of one’s cells. In fact, every single cell in a human body contains the exact same genes; however, the “activity” of genes varies from cell to cell. Between different human bodies and between different species the genes are not the same. Having a tool that identifies and compares genes between two or more samples would enable scientists to understand more about how genes affect who we are, who we aren’t, why we develop a certain disease and why we don’t. Such a tool was one of the outcomes of The Human Genome Project.

Double-stranded DNA (Deoxyribonucleic acid)

The goal of the Human Genome Project “was to provide researchers with powerful tools to understand the genetic factors in human disease, paving the way for new strategies for their diagnosis, treatment and prevention.” ( National Institutes of Health, 2009)Based the development of genomic-scale technologies such as DNA microarrays, one can say that the project reached its goal. A DNA microarrayis a tool that “uses genome sequence information to analyze the structure and function of tens of thousands of genes at a time.” (Bonetta, 2009) Before the invention of DNA microarrays, scientists were limited to the analysis of genes one or two at a time, making the “snap shot” of what was going on with the rest of the genome very limited. So why is this important? DNA microarrays are helping researchers learn more about human diseases, what causes them, how to identify them, and how to treat them. We now know more about complex diseases such as diabetes, multiple sclerosis, heart disease, and cancer that we have ever known before. For some diseases, such as multiple sclerosis, researchers have been able to identify specific genes that influence the risk of getting the disease.(Stimson, 2007)They have found that most diseases that are affected by one’s genes are influenced by many, many genes and not just one or two. Such discoveries may eventually lead to the development of therapeutics that are needed to prevent a disease or to possibly cure it.

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Page 2 of 21 DNA Microarray PK

DNA microarrays are not only used in the medical field, but in other industries such as forensics, agriculture, and toxicology. In this unit you learn about the various applications of the DNA microarray and how this device works. You will also begin to study how microarrays are fabricated. A related activity provides the opportunity to simulate a fabrication process in order to gain a better understanding of these devices – how they work and how they are made. This graphic is a simplification of a DNA Microarray. Fabricated onto a substrate (represented in gold) are single-stranded DNA molecules (probes). These probes are used to identify and recombine (hybridize) to complementary DNA(targets) in the sample being analyzed. We will discuss this process in more detail in this unit.

Objectives  

Describe three applications of the DNA microarray. Explain how a DNA microarray works from hybridization to interpretation.

Terminology(Definitions are provided in the Glossary at the end of this unit) Address Allele Capture molecules cDNA DNA DNA microarray DNA replication Electrophoresis Feature Gene Gene expression GeneChip© Genome Hybridization Microarray


mRNA Nucleotides Oligonucleotide (or oligo) Polymerase chain reaction (PCR) Polymorphisms Reverse transcriptase Reverse transcription RNA RNA polymerase SNPs (single nucleotide polymorphisms) Southern Blot Substrate Target molecules Transcription


Let’s Review the Basics Before jumping into how a DNA microarray works, let’s have a quick review of Deoxyribonucleic acid (DNA), DNA replication and DNA hybridization. If you get stuck on a term, check the glossary at this end of this unit for its definition. A Review of DNA(V. Celeste Carter, 2009) Deoxyribonucleic acid (DNA) is a long polymeric molecule (found in most cells) that functions in the chromosome as the carrier of genetic information. It is a double stranded helix with a uniform diameter. The steps of the helix are formed by base pairs consisting of four (4) nitrogenous bases (Adenine (A), Thymine (T), Guanine (G), and Cytosine (C)). The ladder or rails are formed by nucleotides which are the nitrogenous bases, each consisting of a five-carbon sugar and at least one phosphate group which join the nitrogenous bases along the length of the DNA molecule. The two strands are anti-parallel and demonstrate complementary base-pairing (i.e., A-T, T-A, G-C, and C-G). (see graphic right) The bases are joined in the middle using hydrogen bonds. An A-T and T-A pairing each have two hydrogen bonds while a G-C and C-G pairing each have three hydrogen bonds. The genetic information in the molecule is stored in the linear sequences of the base pairs. For example, A-T, T-A, G-C, and C-G is one partial sequence whereas T-A, G-C, A-T, and T-A may be another sequence. One DNA molecule may consist of millions of base pairs and thus, millions of linear sequences. DNA Transcription(V. Celeste Carter, 2009) DNA transcription is the process that creates a messenger Ribonucleic acid molecule or mRNA. A mRNA is needed to create a copy of a DNA molecule. During transcription the DNA sequences are “copied” into the mRNA molecule as shown in the graphic. In a process called “reverse transcription”, the mRNA sequences are transferred into a DNA copy or cDNA. Transcription is defined as DNA-directed RNA synthesis. It requires a DNA template, RNA polymerase (an enzyme that produces RNA), and ribonucleotide subunits. Transcription produces RNA complementary to the original DNA. This RNA is called the messenger RNA (mRNA). Previously we mentioned a gene’s “activity” and that even though each cell contains the same genes, the activity of those genes vary from cell to cell. A gene’s activity is described as either “on” or “off”. A gene is “on” when it is producing a mRNA (i.e., DNA transcription); otherwise it is “off”. DNA microarrays that create “expression profiles” identify the on/off activity of genes within a cell or organism; however, to do this, a DNA copy (cDNA) of the mRNA must be made. This is called “reverse transcription.” Southwest Center for Microsystems Education (SCME) App_BioMEM_PK24_PG_032514


There are enzymes on the surfaces of your fingers and in every cell that is designed to break down RNA (not the case with DNA). Scientists will take a special enzyme and take that mRNA in cDNA. It is the cDNA that actually gets used. Reverse Transcription DNA microarrays use the cDNA rather than the mRNA because mRNA are unstable and do not last long. Such instability would make a microarray analysis unreliable. cDNA are more stable and thus, more reliable. cDNA are less likely to degrade during the microarray process. In reverse transcription, DNA copies (cDNA) are made from mRNA molecules as shown in the graphic. Reverse transcription provides the cDNA needed for DNA microarrays to study expression profiles or gene activity in cells. DNA Hybridization DNA hybridization refers to the process in which a double-stranded DNA (dsDNA) helix is denatured (or separated) into two, single stranded DNA (ssDNA) molecules by disrupting the hydrogen bonds that hold the two strands together.(See the graphics below.) Since hydrogen bonds are relatively weak, they can be disrupted by simply heating a DNA buffered solution and changing the solution chemistry with extremes of pH and high salt concentrations. The image below illustrates a dsDNA molecule sequence and the hydrogen bonds that hold the two strands of the DNA together at each base-pairing (A-T, T-A, C-G, and G-C).



The graphic below shows the non-covalent hydrogen bonds between the pairs as dashed lines. Left is an A-T base pair with two hydrogen bonds; right is a G-C base pair with three hydrogen bonds.

DNA denaturation is reversible. If the buffer conditions and temperature are slowly changed back to normal, the two strands of ssDNA will again bind to each other, reannealing the two single strands back to their original double-stranded structure. “DNA hybridization” is when the denatured DNA molecules are cooled down in the presence of ssDNA molecules from another source. These DNA molecules from another source are introduced to the original DNA solution during the reannealing step. If the original ssDNA strands have sequences that are complementary to the introduced ssDNA strands, they can form dsDNA hybrid molecules with one strand from each (an original ssDNA and the source ssDNA). The following graphic illustrates the hybridization process.



The ability to make DNA hybrids is used in standard techniques in molecular genetics such as the binding of oligonucleotide (a short nucleic acid polymer, typically with 20 – 50 bases) probes in a Southern blot and the annealing of primers in PCR (polymerase chain reaction).  Southern blot is a technique used to detect a specific DNA fragment in a DNA electrophoresis gel; in other words, to locate a specific base or DNA sequence within an entire genome.  PCR is used to amplify DNA sequences and to make numerous copies of specific DNA segments quickly and accurately. In each case, a synthetic single-stranded oligonucleotide is designed to search and find a complementary DNA sequence, then form a DNA hybrid. This process is similar to the “find” command in a word processing program. If the complementary DNA sequence to be found exists within the page of words, the oligonucleotide (or oligo) locates the word (or cDNA) and tags it. In DNA hybridizations, the oligo strand that carries a label (or sequence) for detecting the base-pairing of a hybrid molecule is called the probe. DNA Hybridization Activity This is a good time to take a break from reading and complete an on-line tutorial on DNA Hybridization. Please complete the DNA Hybridization Activity that is part of this learning module. What is a DNA Microarray? DNA microarrays use gene sequencing and DNA transcription and hybridization to analyze and identify thousands of genes simultaneously. Each microarray consists of hundreds or thousands of gene sequences (ssDNA molecules or oligos) mounted on a chip and used as “probes”. These probes detect complementary DNA (cDNA) fragments or cDNA copied from messenger RNA (mRNA) in a sample. The cDNA are the target molecules (as shown in the graphic). The DNA microarray relies on hybridization of DNA fragments to an oligo DNA sequence (a specific A,C, G, T combination). In DNA microarrays synthetic DNA oligos are fabricated and used as the capture molecules or probes. These synthetic DNA oligos are fabricated onto a solid surface (substrate) before the hybridization step. In the graphic to the right, the probes are the oligos. This graphic illustrates six identical probes on a substrate and several possible targets. Three of the probes have identified cDNA (targets) and have reannealed or are in the process of reannealing into a dsDNA. Those cDNA that do not match this specific probe’s sequence continue to move through the microarray looking for a matching sequence at another location within the array.



In a DNA microarray, a grid is laid out on a surface such as glass microscope slide or silicon substrate, with an array of different DNA sequences. Each position of the array (called an “address” or “feature” of the grid), is looking for a specific gene sequence found in a particular organism. Therefore, each feature has a unique set of synthetic oligo probes attached to it. Each feature may contain hundreds or thousands of identical probes (oligos), while each array may contain tens of thousands of features. Because an array contains thousands of features, it can simultaneously search and “find” thousands of specific genes. To ensure the accuracy of any DNA microarray test, several types of controls may be used (more on this later). In many tests, a control sample is used along with the test sample (both containing millions of cDNA). Detection of cDNA captured (or hybridized) on a microarray is most often done by tagging the cDNA in the control and test samples with a green and red fluorescent dye molecule, respectively. After hybridization, a laser scans the microarray and the presence of specific target DNA is detected by the fluorescence of the label at that position on the microarray. We’ll discuss the function of the control sample as well as the meaning of the different colors (red, yellow, green, and black)in more detail later on in this unit.

This image to the right is of a DNA microarray as seen through a microscope. Each tiny dot corresponds to an address or feature with oligos corresponding to one of the organism's thousands of genes. The color of the dot indicates the relative activity level of a gene. The red dots indicate that the targeted DNA is present in the test sample. [Image courtesy of NASA. Image credit: James Smiley.]



How DNA Microarrays Work This graphic illustrates a DNA microarray prior to being exposed to the control and test sample (or target) genes. The different colored blocks in the graphic (green, blue, yellow, red) represent different features (addresses) in a microarray. The oligos attached at each feature are “ssDNA probes” all having the same sequence (in this feature, G-T-A-C-T-A-G-T-A-C-T-A – bottom to top), of 20 to50 nucleotides, and up to a million probes per feature. Each oligo sequence is complementary to a gene of interest in the control and test samples. DNA microarray expanded from many features (bottom grid) to a few features (middle grid), to a single feature (top grid) depicting a unique DNA sequence (G-T-A-C-T-A…). The coloration in this graphic is strictly to illustrate different locations (features) of ssDNA sequences in a DNA microarray. DNA, and thus a DNA microarray is actually colorless.

DNA Microarray Controls Before moving on, let’s talk about “controls”. There different types of control used in a microarray experiement. Some controls are “positive” and “negative” controls, while others allow for direct comparisons between two different cells – a control cell and a test cell. Positive and Negative controls Positive and negative controls exist in all biological assays (tests that analyze biomolecules in specific events or conditions). The function of positive and negative controls is even more important for DNA microarrays because of the complexity of microarray fabrication. The purpose of positive and negative controls is to verify the overall performance of the microarray (Is it accurate and can it be trusted?) and the analytical technique (Were the samples prepared properly and the procedures executed correctly?). A negative control is an array feature that is designed to have NO binding or hybridization. Its purpose is to avoid getting “false-positives” – positive results that should have been negative. In the design of a DNA microarray, bogus features that have no oligos attached may be interspersed throughout the array. If something does bind to one of these features, then the results of the test should be questioned and the test repeated. Prior to repeating the test, adjustments should be made to the preparation to better ensure accurate results. A positive control is one that you expect to show a positive result. An example is a feature that contains a gene sequence that is ALWAYS present; therefore, you should see hybridization with both the control and test DNA samples. If this positive control does not show hybrization with both samples, then one would have to assume that there may be “false negatives” elsewhere in the array. Therefore, the assay Southwest Center for Microsystems Education (SCME) App_BioMEM_PK24_PG_032514


results should not be trusted. In the case of a “negative” positive control, one should examine the procedure used to prepare the DNA samples to ensure that it was correct. If the procedure was correct, then one should suspect that the microarray is defective. In this case, the test should be discarded. Direct Comparison Controls In this type of control, each feature of the array is a comparison of the test sample DNA to the control sample DNA. Such controls are for the direct comparison of each address (or each gene) between two different cells. Below are examples of DNA microarray testing and direct comparison controls: 1. Testing the effects of drugs or toxins on gene expression (which genes are “active” or “inactive”) – In this test, cDNA from an untreated cells would be compared to the cDNA from treated cells. 2. Testing the mutation of genes in cancer cells – In this test, the genomic DNA (gDNA) from the patient’s noncancerous tissue would be compared to the gDNA from the patient’s cancerous tissue or tumor. The results from such a test can help researchers find new cancer genes as well as help doctors decide how aggressive a patient’s cancer is and thus, the best treatment to prescribe. The following cDNA Microarray Experiment is an example of a direct comparison control assay.



Prior to the DNA hybridization reaction between the probes (on the microarray) and targets, two samples are prepared – the test sample and a control sample (the targets). EachssDNA sequence in the control sample is labeled with a green fluorescent marker, while each ssDNA sequence in the test sample is labeled with a red fluorescent label. When the samples are combined and applied to or “washed over” the microarray, DNA targets in the samples find complementary DNA probes within the microarray and hybridize forming dsDNA hybrids. In the graphic below DNA has been extracted from both the control cell (control sample) and the experimental cell (test sample), transcribed to mRNA which is converted to cDNA through reverse transcription. The cDNA are tagged with their respective fluorescent labels (green for control and red for test sample). The two samples are mixed together and each cDNAin the samples hybridize with a complementary synthetic ssDNA at a specific feature in the microarray.

An example of a cDNA Microarray Experiment. RNA is extracted from two different samples and converted into complementary DNA (cDNA), during which the DNA is labeled with florescent compounds. The two samples are then mixed together for comparison and hybridized to the array. Differences in gene expression are revealed by fluorescent patterns on the array.[Courtesy of "The Science Creative Quarterly". scq.ubc.ca. Artist: Jiang Long}

Now let’s talk about how to retrieve and interpret the results of the hybridization processes that take place during a test.



Interpretation of Microarray Results After hybridization is complete, each feature of a microarray grid is scanned by a green laser and a red laser to detect the presence of both control and test DNA hybridization. As shown below, the scan results in an image of red, green, yellow and black dots or labels. Each color represents the activity or expression level at each address with the test or control sample.  A green label at an address indicates the presence of DNA mostly from the control sample.  A red label at an address indicates the presence of DNA mostly from the test sample.  A yellow appearance at an address indicates an equal amount of target DNA from the control sample and the test sample.  No color or black at an address indicates that neither the control nor the test samples had DNA complementary to that DNA probe’s sequence. A fluorescing DNA microarray showing the results of DNA hybridization between the probe and target DNAs. . When cDNA is prepared from a test sample (red) and from a control sample (green), and both are hybridized to the microarray, the color of the dot indicates the relative activity level of that gene. A green dot shows activity in the control only, red shows activity in the test tissue only, and yellow shows activity in both. Black is the absence of activity from either sample. [Image courtesy of NASA]

The images below show an Agilent Technologies microarray printed on a1” x 3” glass slide format. The image on the right shows the microarray after hybridization and while being scanned with a laser. One can see the fluorescence of hybridized molecules.(Agilent, 2003) [Images courtesy of Agilent Technologies]



An Example of an DNA Microarray Analysis In February of 2007, the U.S. Food and Drug Administration (FDA) approved the marketing of the MammaPrint test – a test that uses DNA microarrays to “predict whether existing cancer will metastasize (spread to other parts of the patient’s body). The reoccurrence of cancer is partly dependent on the activation and suppression of certain genes located in the tumor. Prognostic tests like the MammaPrint (below) can measure the activity of these genes and thus help physicians understand their patient’s odds of the cancer spreading.” MammaPrint was developed by Agendia, in Amsterdam, Netherlands. (FDA, 2007) Beloware the results of a MammaPrint developed in 2006.

This image is the gene expression data matrix of 70 prognostic markers genes from tumors of 78 breast cancer patients hybridized using a DNA microarray referred to as the MammaPrint. “Each row represents a tumor and each column a gene…The metastases status for each patient is shown in the right panel. White indicates patients who developed metastases within 5 years after the initial diagnosis, black indicates patients who continued to be metastasis free for at least 5years.”(Glas, 2006) Those patients above the threshold (yellow line) have a low risk of the cancer spreading to other parts of the body, while those below the line are considered high risk patients and have a poor prognosis. Results such as these and similar DNA microarrays tests provide medical professionals with knowledge of one’s level of susceptibility to a specific disease, leading to possible prevention or treatment, and probable prognosis.



Applications of DNA Microarrays DNA microarrays are being use to study the human genome as well as the genome of other species. They are used for many different purposes and applications. For each species and application a specific selection of probes are included in the array. Researchers can purchase a microarray specific to a particular species’ genome, such as a mouse microarray or a human microarray. Such microarrays contain a probe for each of the genes found in that organism. Genetic comparisons can now be made between organisms of different species or the same species. Two GeneChips® by Affymetrix. One for the Human Genome and one for Mouse Genome. Image courtesy of Affymetrix.

Types of Microarrays There are two basic types of DNA microarrays: direct detection of genes and gene expression. •

Direct detection microarrays detect specific genes or gene mutations within a sample, such as the example above for the BRCA1 mutation. Direct detection microarrays are being used to identify specific genes that cause a specific disease, and to screen for mutations that are responsible for genetic disorders when there are multiple gene mutations that can possibly cause the disorder. Direct detection microarrays are being used to profile somatic mutations in cancer, for forensic applications, genotyping, identifying DNA-based drugs, and in agriculture, “to guide the genetic selection process of milk-producing animals.” (Mertens, 2009) SNP (single nucleotide polymorphisms) chips – a type of direct detection DNA microarray The SNP chips in the photograph are bovine assays (or analysis) that “easily and quickly identify regions within the bovine genome that harbor variants that cause the animals to differ in the outward expression of important traits, allowing scientists to predict an animal’s genetic (Mertens, 2009) merit from its SNP profile.” [Courtesy of Jeremy Taylor, Animal Genomics, University of Missouri]

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Gene expression microarrays detect “expression levels” in a sample - when mRNA copies to cDNA (i.e., which genes are “active” or “inactive”). This information is called a “gene expression profile”. Gene expression microarrays detect how cells and organisms change and adapt to specific stimuli such as changes in the environment or one’s disease state. Let’s remember that every cell in a human’s body contains the same genes. However, the same genes are not active in every cell. Gene expression microarrays allow us to identify which genes are active and which are inactive in different cell types. They also allow us to determine which genes are active in different cell states. For example, gene expression microarrays can identify which genes are turned on during cell division or when exposed to an external stimuli, such as a



drug. This analysis enables “scientists to understand both how these cells function normally and how they are affected when various genes do not perform properly.”(NHGRI, 2009) Gene expression microarrays are being used to study cancer-causing genes and to customize drug therapies based on how our genes react to specific drugs or drug dosages. They are being used to study how cells react to changes in the environment, such as increases in pollutants or toxins. As you can see from this discussion, DNA microarrays are being used to classify cancers, to assess one’s risk for a specific disease, and to track disease progression and predict prognosis. They are proving to be invaluable for identifying drug therapies and drug response (personalized medicine). In genetics, DNA microarrays allow us to compare the genes between two or more different organism as well as the genes within the same organism. Outside of the medical field, DNA microarrays are being used to ensure that our food and the air we breathe are safe, and to assist farmers and ranchers in food production. “If the work involves the DNA or the genetics of a cell, …microarrays can be used.”(Zaccheo, 2005) Key features of DNA Microarrays The key feature of DNA microarrays is the ability to screen for many DNA sequences in one run. In fact, microarrays have enough features (addresses) to screen ALL of the genes of an organism’s genome in one hybridization experiment. Even with thousands upon thousands of different features, the microarray itself is very small - the size of a microscope slide and even smaller. So, although they can store as many as a million features and can screen for the presence of a million different DNA sequences in a single experiment, their size can be on the order of a regular size postage stamp! The size of the individual feature is even smaller – MUCH smaller. Each feature can be as small as 200 nanometers (200 x 10-9 meters, or 0.2 micrometers) – placing this technology in the “nanotechnology” range. The feature size makes fabrication of DNA microarrays a challenge but not impossible since much of the technology that is needed to fabricate microarrays already exists. Many of the fabrication methods for microarrays come from semiconductor fabrication technology as well as microtechnology. For example, one type of microarray fabrication technique uses a printer similar to an ink-jet printhead (a micro-size device) to print the addresses onto the microarray slide. However, the “ink” is an oligonucleotide solution. Another method of microarray fabrication uses the photolithography process used in micro-fabrication. Let’s take a closer look at both processes.



DNA Microarray Fabrication The DNA microarrayer (below left) “prints” oligonucleotide probes to specific addresses on a microscope slide using the microtechnology of an ink-jet printer (below right). The inkjet “printhead” consists of an ink reservoir and piezoelectric actuators that allow the fluid ink to flow from the reservoir to the nozzles. Because the diameter of the nozzles is in the micrometer-scale (between 1 to 100 micrometers), capillary action moves the molecules of ink through the tiny nozzles. This same technology is used to achieve the nano-size probes when printing microarrays. Because the size of a probe is in the nano-scale, the printhead nozzles are smaller allowing a single microarray to have as many as a million features printed onto one slide.

Alternatively, the Affymetrix GeneChip® technology uses a fabrication technique called photolithography to synthesize the oligonucleotides on a silica (or “chip”) surface. The Affymetrix GeneChip® is a DNA microarray that has oligonucleotide probes synthesized in place on a silicon chip using a photolithography process borrowed from the semiconductor fabrication industry. The oligonucleotides on these arrays tend to be shorter, generally 20 nucleotides long and the array is miniaturized even more than those found in other DNA microarrays. Each feature on a GeneChip® may be as small as 50 nanometers square – almost 2000 times smaller than the width of a strand of hair! A more in-depth look at this process is included in the DNA Microarray Model Activity. [The photo shows a GeneChip® (blue) for the human genome. The case that encloses the GeneChip® is about as wide as the length of a wooden match. Image courtesy of Affymetrix] The next generation of DNA microarrays will be MEMS devices (microelectromechanical systems) containing mass sensitive microcantilevers and electrochemical detection. These arrays will be 100 times more sensitive, down to attomolar (10-18) sensitivity! They will also have greater precision and better reproducibility, faster and more robust (fewer steps in assay).(Yan, Tang, Zhai, & Ju, 2007)



Summary The DNA microarray has opened up a whole new frontier for exploration in medical research, drug development, forensics, toxicology, and food production, just to name a few. All of the information derived from DNA microarrays affects us all in one way or another. Through the hybridization of synthetic oligos and target DNA molecules we can identify the presence of specific genes, mutation and pathogens. We are getting closer and closer to knowing what makes us tick, what makes us sick and what can make us well. Food for Thought The DNA microarray may be small, but when it comes to career potential, it’s huge!  Discuss at least three applications or potential applications of DNA microarrays.  How does a DNA microarray identify a target DNA?  What are some of the careers that one might look into that involve the use of or the fabrication of DNA microarrays?

References 1. National Institutes of Health. (2009, March 11). Human Genome Project Fact Sheet. Retrieved from National Human Genome Research Institute: http://www.nih.gov/about/researchresultsforthepublic/HumanGenomeProject.pdf 2. Agilent. (2003, October 8). Image Library. Retrieved from Agilent Technologies: http://www.agilent.com/about/newsroom/lsca/imagelibrary/index_2003.html 3. Bonetta, L. (2009). The Basics of DNA Microarrays. Retrieved from BioInteractive - Howard Hughes Medical Institute: http://www.hhmi.org/biointeractive/genomics/microarray.html 4. Cooper, C. S. (2001, March 20). Applications of microarray technology in breast cancer research. Retrieved from Breast Cancer Research: http://breast-cancerresearch.com/content/3/3/158 5. Cooper, C. S. (2001). Applications of microarrya technology in breast cancer research. Retrieved from Breast Cancer Research: http://breast-cancer-research.com/content/3/3/158 6. FDA. (2007, February 6). FDA Clears Breast Cancer Specific Molecular Prognostic Test. Retrieved from FDA-News & Events: http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncement/2007/ucm108836.htm 7. Glas, A. M. (2006). Converting a breast cancer microarray signature into a high-throughput diagnostic test. Retrieved from BioMed Central: http://www.biomedcentral.com/14712164/7/278 8. Lettieri, T. (2006, January). Environmental Health Perspectives. Retrieved from PubMed Central: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1332648/ 9. Mertens, R. (2009, March 15). A new chip identifies important bovine genomic traits. Retrieved from College of Agriculture, Food and Natural Resources: http://cafnr.missouri.edu/news/stories2009/snp-chip.php 10. NCBI. (2001). Appendix I. Immunologists' Toolbox (Immuno Biology). Retrieved from National Center for Biotechnology: http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=imm&part=A2395 Southwest Center for Microsystems Education (SCME) App_BioMEM_PK24_PG_032514


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NCBI. (2007, July 27). Microarrys: Chipping Away at the Mysteries of Science and Medicine. Retrieved from NCBI (National Center for Biotechnology Information): A Science Primer: http://www.ncbi.nlm.nih.gov/About/primer/microarrays.html NCI. (2010). Dictionary of Cancer Terms. Retrieved from National Cancer Institute: http://www.cancer.gov/dictionary/?CdrID=386201 NHGRI. (2009, December 17). DNA Microarray Technology. Retrieved from National Human Genome Research Institute: http://www.genome.gov/10000533 Stimson, D. (2007, July 29). After a decades-long search, scientists identify new genetic risks factors for multiple sclerosis. Retrieved from EurekAlert!: http://www.eurekalert.org/pub_releases/2007-07/nion-aad072607.php V. Celeste Carter, P. D. (2009). DNA Overview Learning Module. Retrieved from Southwest Center for Microsystems Education: http://scmenm.net/scme_2009/index.php?option=com_docman&task=cat_view&gid=119&Itemid=53 Wikipedia. (2010, May 11). DNA Microarray. Retrieved from Wikipedia, the Free Encyclopedia: http://en.wikipedia.org/wiki/DNA_microarray Yan, J. W., Tang, J., Zhai, C., & Ju, H. (2007). A Disposable Simultaneous Determination of Tumor Markers. Clinical Chemistry 53:81 , pp. 1495-1502. Zaccheo, R. K. (2005). GeneChip Microarrays Curriculum for Life Science Teachers. Retrieved from IISME: http://www.iisme.org/etp/HS%20Science-%20GeneChip%20Mciroarrays.pdf



Glossary Address – A single location on a DNA microarray that can contain up to a million of the same ssDNA sequence. Also called a feature. Allele – One member of a pair or variation of genes that occupy a specific position on a specific chromosome. Anti-parallel – Two molecules that are side-by-side by run in opposite directions. In DNA the two strands are anti-parallel because the head of one strand lays against the tail of the other strand. Base Pair – The pairing of a nitrogenous base using two hydrogen bonds for A-T and T-A pairs and three hydrogen bonds for C-G and G-C pairs. Capture molecules – Specific DNA binding sequences used as probes on DNA microarrays. cDNA (Complementary DNA) - Single-stranded DNA that is complementary to messenger RNA or DNA that has been synthesized from messenger RNA by reverse transcriptase. Deoxyribonucleic acid (DNA) - A long linear polymer formed from nucleotides pairs, and shaped like a double helix; associated with the transmission of genetic information. DNA microarray - A tool that “uses genome sequence information to analyze the structure and function of tens of thousands of genes at a time.” A microchip that holds DNA probes that form half of the DNA double helix (called single-stranded DNA or ssDNA) and can recognize other ssDNA from samples being tested. DNA replication – The process in which each strand of a double-stranded DNA molecule serves as template for the reproduction of two identical DNA molecules. Electrophoresis- A method used to separate particles, such as DNA or proteins, in which an electric current is passed from one electrode to another of an opposite charge through a medium, and the separation of the molecules depends on the rate at which the molecules travel towards the electrode based on their electrical charge. Jonas: Mosby's Dictionary of Complementary and Alternative Medicine. (c) 2005, Elsevier. – The movement of charged suspended particles through a liquid medium in response to changes in an electric field. Mosby's Medical Dictionary, 8th edition. © 2009, Elsevier. Feature - A single location on a DNA microarray that can contain up to a million of the same ssDNA sequence. Also called an address. Gene - A length of DNA sequence that contains information that is capable of being translated into a polypeptide product. Gene Expression – Conversion of the information encoded in a gene first into a messenger RNA and then to a functional gene product, which is often a protein.



GeneChip®- A DNA microarray developed by Affymetrix that uses ssDNA probes to recognize complementary ssDNA from samples being tested Genome – An organism’s genetic material which is made up of molecules of DNA and found in the nucleus of eukaryotic cells and in the cytoplasm of prokaryotic cells. Hybridization–A process of combining two complementary single-stranded DNA into a single doublestranded molecule through base pairing. Microarray– A micro-sized lab-on-a-chip (LOC) with a two-dimensional array printed onto a substrate. The array is used to assay (analyze) large amounts of biological materials in a sample solution. Nucleotides – Molecular subunits consisting of a nitrogenous base (A, T, C, or G), a sugar and at least one phosphate, that when joined together, make up the structural unit of RNA and DNA. Oligonucleotide – A short fragment of single-stranded DNA typically 5 to 50 nucleotides long. http://www.ncbi.nlm.nih.gov/About/primer/microarrays.html> Polymerase Chain Reaction (PCR) -A laboratory technique that can amplify the amount of DNA from a tiny sample to a large amount within just a few hours. PCR can take one molecule and produce copies of a DNA sequence through replication with a heat-stable DNA polymerase and two oligonucleotide primers that flank the target sequence. Polymorphisms - Variations of forms. A DNA polymorphism is a mutation or change in DNA sequence that is found in at least 1% of the species. Reverse transcriptase – An enzyme isolated from certain viruses that can make a DNA sequence complementary to an RNA sequence. The copy DNA sequence is referred to as cDNA. Reverse transcription– The process of making a copy DNA sequence from an RNA sequence using the enzyme reverse transcriptase. Ribonucleic acid (RNA) -A polymer consisting of a long, usually single-stranded chain of alternating phosphate and ribose units with the bases bonded to the ribose. RNA polymerase– The enzyme responsible for locating a gene on a DNA sequence and making an RNA copy from the DNA sequence of the gene. Southern Blot – The transfer of DNA fragments in an electrophoresis gel to a plastic membrane, followed by hybridization with a radio labeled oligonucleotide probe complementary to specific DNA sequences. Used in DNA fingerprinting. Single Nucleotide Polymorphisms (SNPs) - Microarrays developed to locate genetic variations (i.e., sequence polymorphisms – the difference between the queen bee, worker bee and drone) within the same organism or species are mapped for a particular organism. Target molecules–The molecules in a sample solution that are to be assayed or analyzed using a microarray. Transcription– The processEducation of making RNA sequences from DNA sequences, using RNA polymerase Southwest Center for Microsystems (SCME) Page 20 of 21 enzyme. App_BioMEM_PK24_PG_032514 DNA Microarray PK

Disclaimer The information contained herein is considered to be true and accurate; however the Southwest Center for Microsystems Education (SCME) makes no guarantees concerning the authenticity of any statement. SCME accepts no liability for the content of this unit, or for the consequences of any actions taken on the basis of the information provided.

Support for this work was provided by the National Science Foundation's Advanced Technological Education (ATE) Program. This Learning Module was developed in conjunction with Bio-Link, a National Science Foundation Advanced Technological Education (ATE) Center for Biotechnology @ www.biolink.org.



DNA MicroarrayActivity: DNA Hybridization Participant Guide Description and Estimated Time to Complete This activity provides the instructions for accessing an on-line tutorial on DNA Hybridization. DNA Hybridization is the process used to identify the degree of genetic similarity between pools of DNA. DNA Microarrays depend on the hybridization between single-stranded DNA (ssDNA) probes and ssDNA copies in control and test samples. In order to get the most from this activity, you should have basic understanding of the DNA molecules, its four (4) nitrogenous bases (Adenine (A), Thymine (T), Guanine (G), and Cytosine (C)) and DNA base pairs (i.e., A-T, T-A, G-C, and C-G), which are the building blocks of DNA. (Refer to the References section for review sources.) After completing the tutorial, this activity provides post-activity questions that allow you to demonstrate your understanding of the information presented in the tutorial. Estimated Time to Complete Allow at least 30 minutes to complete.

Introduction to DNA Hybridization The ability to make DNA hybrids through DNA hybridization is used in standard techniques in molecular genetics such as the binding of oligonucleotide (a short nucleic acid polymer, typically with 20 – 50 bases or nucleotides) probes in a Southern blot and the annealing of primers in PCR (polymerase chain reaction).  Southern blot is a technique used to detect a specific DNA fragment in a DNA electrophoresis gel; in other words, to locate a specific base or DNA sequence within an entire genome.  PCR is used to amplify DNA sequences and to make numerous copies of specific DNA segments quickly and accurately. In each case, a synthetic single-stranded oligonucleotide is designed to search and find a complementary DNA sequence, then form a DNA hybrid. This process is similar to the “find” command in a word processing program. If the complementary DNA sequence to be found exists within the page of words, the oligonucleotide (or oligo) locates the word (or complementary DNA) and tags it. In DNA hybridizations, the oligo strand that carries a label (or sequence) for detecting the base-pairing of a hybrid molecule is called the probe.

The process of hybridization is similar to DNA replication. Hybridization refers to the process in which a double-stranded DNA (dsDNA) helix is denatured (or separated) into two, single stranded DNA (ssDNA) molecules by disrupting the hydrogen bonds that hold the two strands together.(See the graphics below.) Since hydrogen bonds are relatively weak, they can be disrupted by simply heating a DNA buffered solution and changing the solution chemistry with extremes of pH and high salt concentrations. The image below illustrates a dsDNA molecule sequence and the hydrogen bonds that hold the two strands of the DNA together at each base-pairing (A-T, T-A, C-G, and G-C).

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The graphic below shows the non-covalent hydrogen bonds between the pairs as dashed lines. Left is an A-T base pair with two hydrogen bonds; right is a G-C base pair with three hydrogen bonds.

DNA denaturation is reversible. If the buffer conditions and temperature are slowly changed back to normal, the two strands of ssDNA will again bind to each other, reannealing the two single strands back to their original double-stranded structure. “DNA hybridization” is when the denatured DNA molecules are cooled down in the presence of ssDNA molecules from another source. These DNA molecules from another source are introduced to the original DNA solution during the reannealing step. If the original ssDNA strands have sequences that are complementary to the introduced ssDNA strands, they can form dsDNA hybrid molecules with one strand from each (an original ssDNA and the source ssDNA). The following graphic illustrates the hybridization process.



Activity Objectives and Outcomes Activity Objectives  Explain how probes made of nucleotide sequences bind to complementary target DNA sequences.  State at least two bioMEMS applications that study or utilize DNA hybridization. Activity Outcomes You will use the information gathered in this tutorial to explain how DNA hybridization occurs and its applications for bioMEMS. Resources Computer with high-speed Internet access. Documentation Your documentation should include all of the questions asked during each stage of this activity and your answer to each of these questions. Documentation should also include the Post-Activity Questions and your answers.



Activity: DNA Hybridization This activity will help you better understand the process of DNA hybridization and how it applies to bioMEMS applications. You will utilize the tutorial at The Molecular Workbench. 1. Go to The Molecular Workbench at http://workbench.concord.org/database 2. In the upper right corner "Jump to Activity" # 265. Select "Student". This should take you to an interactive called DNA Hybridization. (NOTE: If you have a problem with the link, do a search within Molecular Workbench for DNA Hybridization.) 3. Launch Activity (It may take a few minutes to download.) 4. Watch the modeling simulation. 5. When it stops and says "Probe Target Found", take a snapshot and describe the image you see. Record a sketch of the image and your description in this activity's documentation. 6. Resume simulation. Watch for a few more minutes and record what happens. 7. In the bottom left corner of the screen select Southern Blot. There are 5 parts to this tutorial. Complete all five starting with “Introduction”. During this activity, record all questions and your answers for your documentation. 8. Use the “Back” button to return to the “Modeling DNA Hybridization” page or Copy and paste this URL (http://mw2.concord.org/public/student/lab/hybridization.cml) in the Molecular Workbench address box and RELOAD. 9. Select DNA double-helix (bottom left-hand corner). Use your cursor to rotate the molecule. Right click and change the “style” of the DNA double-helix in order to study it in different forms. 10. Answer the Post-Activity questions at the end of this activity.



Post-Activity Questions 1. What is the Southern Blot? 2. What is a nucleotide? 3. DNA hybridization makes use of base pairing between __________________________________________________________

4. Where is DNA hybridization currently found in bioMEMS technology? (State at least two applications) 5. What is another possible application of bioMEMS and DNA Hybridization?

Summary Being able to see and study the DNA hybridization process has become a reality. BioMEMS devices are being designed to use this process for diagnostics and therapeutic applications. References 1 The Molecular Workbench at http://mw.concord.org/(Program funded by the National Science Foundation) 2 SCME's BioMEMS Applications Overview Primary Knowledge unit 3 SCME’s DNA Overview Learning Module 4 SCME’s DNA Microarray Primary Knowledge unit

Support for this work was provided by the National Science Foundation's Advanced Technological Education (ATE) Program. This Learning Module was developed in conjunction with Bio-Link, a National Science Foundation Advanced Technological Education (ATE) Center for Biotechnology @ www.bio-link.org.



DNA Microarray Terminology Activity Participant Guide Description and Estimated Time to Complete In this activity you demonstrate your knowledge and understanding of the terminology associated with DNA (Deoxyribonucleic acid) microarrays – their applications, operation, interpretations, and fabrication. If you have not reviewed the unit DNA Microarrays, you should do so before completing this activity. This DNA Microarray unit provides the background information needed to best understand the terms and concepts presented in this activity. This activity consists of a crossword puzzle, matching table, and Post-Activity Questions that provide a better understanding into the world of DNA Microarrays. You are to complete the crossword puzzle OR the matching table, AND the Post-Activity questions. You could do all three if you choose. Estimated Time to Complete Allow at least two hours to complete this activity. Introduction In order to understand DNA Microarrays, you need to have an understanding of DNA, RNA (Ribonucleic acid), DNA transcription and reverse transcription, and hybridization. WOW – now that’s a mouth full.  Deoxyribonucleic acid (DNA) is a long polymeric molecule (found in most cells) that functions as the carrier of genetic information.  Transcription produces a RNA complementary to the original DNA when a double-stranded DNA (dsDNA) divides. This RNA is called the messenger RNA (mRNA).  Reverse transcription makes a copy DNA from the mRNA. The cDNA is used by DNA microarrays in the target samples.  DNA hybridization is when denatured DNA molecules are cooled down in the presence of ssDNA molecules from another source. If the original ssDNA strands have sequences that are complementary to the introduced ssDNA strands, they can form dsDNA hybrid molecules with one strand from each (an original ssDNA and the source ssDNA). To understand the DNA microarray, one must understand the terminology associated with it; otherwise, it’s like navigating a new city without a map – you get lost.

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Activity Objectives and Outcomes Activity Objectives  

Identify the specific term associated with a definition or statement related to DNA and DNA microarrays. Explain the relationship between this terminology and the understanding of how a DNA microarray works and is fabricated.

Activity Outcomes At this end of this activity you will have a better understanding of DNA microarray terminology and the various concepts related to DNA microarrays. Resources SCME DNA Microarray Primary Knowledge unit Documentation  Completed crossword puzzle or matching table.  Answers to the Post-Activity Questions. NOTE: Be sure to include ALL sources and references to data and graphics when applicable.



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Questions Answers ACROSS 6. A micro-sized lab-on-a-chip (LOC) with a two-dimensional array printed onto a substrate. The array is used to assay (analyze) large amounts of biological materials in a sample solution. 7.

In a DNA molecule, the order of the nucleotide bases.

10. ssDNA molecules on a DNA microarray used to capture target molecules in a sample. 12. A type of DNA microarray used to detect a specific gene, gene mutations, or deletions of chromosomal DNA. 16. Expression profiles identify the on/off _______________ of genes within a cell or organism. 19. Base ____________ consist of the pairing of complementary nitrogenous bases and form the steps of the DNA helix. 20. A length of DNA sequence that contains hereditary information or an organism. 21. The molecules in a sample solution that are to be assayed or analyzed using a microarray. 22. A blot or a technique used to detect a specific DNA sequence in a DNA sample, to locate a gene within an entire genome. 24. Gene ________________ microarrays measure cDNA from many different genes at the same time. Can be used to study how cells react to external changes. 28. The process of creating a RNA copy from a DNA sequence.

DOWN 1.

When analyzing a DNA microarray result, the color that indicates no activity between the probes and the targets in either control or test sample.

2.

When analyzing a DNA microarray result, the color that indicates activity between the probes and the control sample ONLY.

3.

A long linear polymer formed from nucleotide pairs and associated with the transmission of genetic information.

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A process of combining two complementary ssDNA, from two different sources, into a single dsDNA molecule.

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An address in a DNA microarray containing thousands of the same ssDNA probe molecules.



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Molecules, that when joined together, make up the structural unit of RNA and DNA.

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When analyzing a DNA microarray result, the color that indicates activity only between the probes and the targets from the test sample.

11. Sugar and ______________ groups form the ladder or rails of the DNA helix. 13. A molecule consisting of a long, usually single-stranded chain of nucleotides with the bases adenine, guanine, cytosine, and uracil. 14. The two strands of a DNA molecule are held together by ____________________ bonds. 15. The process in which each strand of a double-stranded DNA molecule serves as template for the reproduction of two identical DNA molecules. 17. When analyzing a DNA microarray result, the color that indicates probe-target activity between both the control and test samples. 18. To change the molecular structure and characteristics of a molecule by chemical or physical means. Also to divide a dsDNA into two ssDNAs. 20. An organism’s genetic material which is made up of molecules of DNA and found in the nucleus of eukaryotic cells and in the cytoplasm of prokaryotic cells. 23. A gene expression _____________ is developed from information about all messenger RNAs (mRNAs) that are made in various cell types. 25. A laboratory technique that can amplify the amount of DNA from a tiny sample to a large amount within just a few hours. 26. The abbreviated form of the term that represents a short fragment of single-stranded DNA typically 5 to 50 nucleotides long. 27. ssDNA that is complementary to a mRNA that has been synthesized by reverse transcription. 29. A change in a single nucleotide in the DNA sequence or when two DNA sequences are identical except for one nucleotide (e.g., A-C-T-C-A-G and A-C-G-C-A-G)



DNA Terminology Match Activity: Match the following terms to their definitions. Answer

Terms

Definitions

Genes

A. A process of combining two complementary ssDNA from different sources into one dsDNA

Alleles

B. A process in which ultra violet light and masks are used to fabricate micro-size devices.

Gene Sequence

C. A control that uses each feature in an array to make a comparison of the DNA in the test and control samples.

Nitrogenous bases

D. When a gene’s mRNA copies to cDNA.

A Nucleotide

E. A short nucleic acid polymer, typically 20 – 50 nucleotides long.

mRNA

F. The use of UV light to degrade the blocking agent on top of a nucleotide base in the feature of an array.

Reverse Transcription

G. One member of a pair or variation of a gene that occupy a specific position on a specific chromosome.

cDNA

H. A base with a sugar and at least one phosphate

Hybridization

I.

Oligonucleotide

J. An address of a microarray that contain hundreds or thousands of the same oligonucleotide.

DNA hybrid

K. The basic biological unit of heredity.

Gene expression arrays

L. When a base attaches to a deprotected base in a feature of a DNA microarray.

Microarray feature

M. Arrays that detect the “expression levels” in a sample – when mRNA copies to cDNA.

Positive and negative control

N. A molecule created during DNA transcription

Direct comparison control

O. A patterned component used to identify specific features that are to be deprotected with UV light during microarray fabrication.

Direct detection arrays

P. Order of base pairs in a DNA segment: A-T, T-A, C-G, A-T, C-G, G-C, T-A

Photolithography

Q. A dsDNA made of ssDNA from two different sources

Deprotect (microarray fabrication)

R. The addition of a blocking agent to the nucleotide bases used to build the oligos of a DNA microarray.

Protect (microarray fabrication)

S. A ssDNA that is a copy of a mRNA

Addition (microarray fabrication)

T. Adenine, thymine, Guanine, and Cytosine

Mask

U. A microarray that detects specific genes or gene mutations within a sample.

In a microarray, the type of controls used to verify the overall performance of the array and analytical technique.



Post-Activity Questions 1. Below is a graphic illustrating a single feature of a DNA microarray.Using DNA and DNA microarray terminology, explain each element (i.e., base, ssDNA, dsDNA, probes, and targets) of the graphic and what “action” is and is NOT taking place.

2. Using the terminology associated with DNA microarrays, explain what is being illustrated in these pictures and what the various squares, dots and colors represent.



3. Read the article linked below andanswer the following: a. Briefly explain how DNA microarrays are being used in this applcation. b. Why does the presence of a specific gene NOT guarantee anything? In other words, if a person has the ACTN3 gene for “explosive bursts of strength”, why doesn’t the presence of the gene guarantee that the person will excel in that ability? c. What other athletic abilities do you think might be genetic (passed down from one generation to another)? Could gene test tell if kids could be sports stars? (Lindsey Tanner, Associated Press, March 8, 2011) (Link: http://www.biosciencetechnology.com/News/FeedsAP/2011/03/industries-could-gene-tests-tell-if-kidscan-be-sports-stars/?et_cid=1245613&et_rid=45505028&linkid=http%3a%2f%2fwww.biosciencetechnology.com%2fNews%2fFee dsAP%2f2011%2f03%2findustries-could-gene-tes )

Summary DNA microarrays use synthetic ssDNA (oligonucleotides) fabricated on a substrate to identify specific genes or DNA sequences in a sample (direct detection) or to identify the activity of DNA in a sample (gene expression profiling). DNA microarrays use DNA transcription and reverse transcription to make the copy ssDNA molecules used in the control and test samples for DNA microarray testing.




DNA Microarray Model Activity Participant Guide Description and Estimated Time to Complete In this activity you study how DNA (Deoxyribonucleic acid) microarrays are fabricated using a photolithography process developed by the semiconductor manufacturing industry. You then apply this knowledge to building a macro-size DNA microarray model. A second activity allows you to simulate the process of matching probes to targets and interpreting the results. These activities should improve your understanding of how DNA microarrays are made and how they identify complementary single-stranded DNA (ssDNA) in a sample. A bonus activity takes the DNA microarray fabrication process one step further and asks you to design a 4 x 4 microarray, identifying the DNA sequence for each feature, and then designing the masks for microarray fabrication. If you have not reviewed the unit DNA Microarrays, you should do so before completing this activity. This DNA Microarray unit provides the background information needed to best understand the concepts presented in this activity. Estimated Time to Complete Allow at least two hours to complete this activity. Introduction DNA microarrays are helping researchers learn more about human diseases, what causes them, how to identify them, and how to treat them. We now know more about complex diseases such as diabetes, multiple sclerosis, heart disease, and cancer than we have ever known before. For some diseases, such as multiple sclerosis, researchers have been able to identify specific genes that influence the risk of getting the disease.(Stimson, 2007) They have found that most genetic disorders are influenced by many, many genes and not just one or two. Such discoveries may eventually lead to the development of the therapeutics needed to diagnose and prevent a disease or to possibly cure it.

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DNA microarrays use gene sequencing and DNA transcription and hybridization to analyze and identify thousands of genes simultaneously. Each microarray consists of hundreds or thousands of gene sequences (or ssDNA molecules) which are mounted on a chip and used as “probes”. These probes detect complementary DNA fragments or cDNA copied from messenger RNA (mRNA) in a sample. The cDNA are the target molecules (as shown in the graphic). The DNA microarray relies on hybridization of ssDNA fragments to an oligonucleotide (oligo) DNA sequence (a specific A,C, G, T combination). In DNA microarrays synthetic DNA oligos are fabricated and used as the capture molecules or probes. These synthetic DNA oligos are fabricated onto a solid surface (substrate) before the hybridization step. In the graphic to the right, the probes are the oligos. This graphic illustrates six identical probes on a substrate and several possible targets. Three of the probes have identified cDNA (targets) and have reannealed or are in the process of reannealing into a double-stranded DNA or dsDNA (i.e., hybridization). Those cDNA that do not match this specific probe’s sequence continue to move through the microarray looking for a matching sequence at another address or feature within the array.

Activity Objectives and Outcomes Activity Objectives  

Using the components provided in a SCME DNA Microarray kit, build a macro-size DNA microarray with at least a 3 nucleotide sequence using the photolithography process developed by Affymetrix for the GeneChipTM. Outline and explain the fabrication steps for an oligonucleotide array.

Activity Outcomes At the end of this activity, you will be able to answer the following questions:  How are oligonucleotides used in a DNA microarray?  How are synthetic oligonucleotides fabricated on a DNA microarray?  How does a DNA microarray identify different target molecules simultaneously?



Resources (Can be downloaded from scme-nm.org – Educational Materials) SCME DNA Microarray Primary Knowledge unit SCME DNA Microarray Model Kit DNA Microarray Fabrication Many of the fabrication methods for microarrays come from semiconductor fabrication technology as well as microtechnology. For example, one type of microarray fabrication technique uses a printer similar to an ink-jet printhead (a macro-size device with micro-size elements called nozzles) to print the addresses onto the microarray slide. However, the “ink” is an oligonucleotide solution. Another method of microarray fabrication uses the photolithography process used in micro-fabrication. Let’s take a closer look at both processes. The DNA microarrayer (below left) “prints” oligonucleotide (oligo) probes to specific addresses on a microscope slide using the microtechnology of an ink-jet printer (below right). The inkjet “printhead” consists of an ink reservoir for each color and piezoelectric actuators that allow the fluid ink to flow from the reservoir to the nozzles. Because the diameter of the nozzles is in the micrometer-scale (between 1 to 100 micrometers), capillary action moves the molecules of ink through the tiny nozzles. This same technology is used to achieve the nano-size probes when printing microarrays. Because the size of a probe is in the nano-scale, the printhead nozzles are smaller allowing a single microarray to have as many as a million to two million addresses printed onto one slide. As the printhead moves across the array, actuation of the piezoelectric crystal causes the prepared oligos to be delivered through the nano-size nozzles to the slide surface without physical contact.

Take a break and watch a video showing the robotic printing of DNA microarrays: “Diagnostic Microarrays” - http://www.youtube.com/watch?v=8hB0hy6_oKg&feature=related



The printing of a DNA microarray is very laborious due to all of the oligonucleotide “inks” that must be prepared for each of the addresses on the microarray. These oligos may be synthesized by chemical methods, one at a time, or they can be prepared enzymatically by a reverse transcription of mRNA isolated from cells to copy DNA or cDNA. Both of these methods of preparing oligo probes from each address of a DNA microarray are long procedures and require a long time to prepare before you have all the sequences needed to print onto a microarray. Alternatively, the photolithography method uses the photolithography process borrowed from the semiconductor fabrication industry in combination with chemical reactions to synthesize oligonucleotides probes on a silicon surface. The Affymetrix GeneChip® (image right) is a DNA microarray, but more specifically an oligonucleotide array that has thousands of synthetic oligonucleotide (oligo) probes fabricated on a silicon chip. The oligos on these arrays are generally 20 nucleotides long and the array itself, is smaller than other types of DNA microarrays. Each address on a GeneChip® may be as small as 50 nanometers (nm) square – almost 2000 times smaller than the width of a strand of hair! Even smaller are the oligos themselves. Each address may contain 11 to 16 DNA copies of the same gene meaning 11 to 16 oligos per 50 nm square address! (Zaccheo, 2005) DNA microarray (2008) allows for assay of approximately 500,000 polymorphisms in a single genome. [Image courtesy of Affymetrix]

In this activity you will simulate the GeneChip® fabrication process for a Oligonucleotide Array which is patented by Affymetrix. So let’s take a more in-depth look into that process.



Fabrication of a Oligonucleotide Array As previously mentioned, oligonucleotide microarray fabrication uses the photolithography process developed from the semiconductor industry and chemical reactions to construct synthetic oligonucleotides onto a silicon substrate. In this approach oligos of different sequences are built from the bottom up in each address of the GeneChipTM by repeating a three step process –Protect , Deprotect, Addition. Each cycle of the three step process adds a new nucleotide base (i.e., A (adenine), T (thymine), G (guanine), or C (cytosine)) to the sequence, allowing the oligos to be built up one nucleotide at a time. Let’s take a look at this process. Initially, a light-sensitive blocking chemical or agent is washed over the entire silicon wafer (one wafer contains hundreds of microchips or microarrays). The blocking agent protects the “surface” of the wafer. In subsequent steps, the blocking agent attaches to the nucleotides in the Protect step of the process. Deprotect (Photolithography) Deprotect uses photolithography which requires a patterned mask and ultraviolet (UV) light to “deprotect” select features of the microarray. Each exposed feature is a location for the addition of the next nucleotide base in a specific sequence. The graphic illustrates the UV light, mask and substrate. Each square in the mask is a select feature or address in the array. The light that travels through the mask degrades the chemical blocking agent, “deprotecting” the areas exposed to UV. Addition- After “deprotect” the wafer is washed with a solution of the specific nucleotide base being added at that step. The nucleotide bases in the solution attach through a chemical reaction to the deprotected areas on the wafer. The blocking agent protects all of the features that were not exposed to the UV light, preventing the addition of a nucleotide base in those features. Protect Once the exposed areas have increased in size by one nucleotide base, a photosensitive blocking or “protective” agent is added to “protect” the new nucleotide bases on the array. The microarray is now ready for another mask or “deprotect” step. Take a few minutes and watch the animation “GeneChip” on YouTube. This animation illustrates the three step process described above. (NOTE: The animation uses “filter” in place of “mask”.) http://www.youtube.com/watch?v=V8uNJCO7Qqo



As seen in the animation, this three step cycle is repeated until the desired oligonucleotides are fabricated throughout the array. For each cycle of the process a mask with a unique pattern is used. An array with 25 nucleotide probes may require as many as 100 masks and thus 100 cycles of the deprotect, addition, and protect process. Therefore, since a new mask is required for each deprotect step of a cycle, the component that ultimately controls the building of ssDNA probes (oligos) on selected positions in the array is the mask. With the first few masks, most of the deprotected areas “link” a nucleotide directly to the substrate. The other deprotected areas attach the nucleotide to a deprotected nucleotide already linked to the substrate. In order to construct an array with specific nucleotide sequences in each feature, each mask is unique and each mask deals with one and only one nucleotide at a time. For example, the following four graphics illustrates the first two masks of a process and the outcome at the end of a three step cycle. Mask 1 (a) identifies 16 features (addresses) that start with the A nucleotide base as the first nucleotide in the sequence. The (b) graphic is the result. The yellow dots represent the A nucleotide that has linked to the substrate. Mask 2 (c) identifies 20 features for the C nucleotide (16 attached to the base and 4 attached to an existing A nucleotide). The (d) graphic is the result. The red dots represent the C nucleotide.

Mask 1 indicates “A” as the first nucleotide in the sequence (in yellow) in columns 1 and 2. Mask 2 indicates “C” as the first nucleotide in the sequence (in red) in columns 3 and 4. However, as you can see in figures (c) and (d), column 2, rows A, C, E, and G, a sequence of AC is indicated. Mask 2 allowed “C” to synthesize onto the deprotected base “A”. (Note: C actually is “on top of” A, not side by side as shown.) This indicates that in these four addresses the first two bases of the oligo sequence is A-C. Each subsequent mask controls the rapid synthesis of the oligo probes by allowing each probe’s sequence to pick up one more link in the chain (i.e., a sequence of A-C-T could become A-C-T-C). “Computer algorithms calculate the optimal design of the masks that will minimize the number of reactions needed.” (Antler, 2004) So putting things in perspective – in the previous example, after two masks, we’ve only just begun with half of the array. As previously mentioned, if we want an array of 100 oligonucleotides, then we’ll need at least 100 masks!



Here’s another presentation by Affymetrix, the developer of this process. 

DNA Chips and microarrays – A UTube video by Affymetrix on the DNA microarray. http://www.youtube.com/watch?v=ui4BOtwJEXs&feature=related

Are you ready? Let build a model of a DNA microarray simulating the process used to fabricate Oligonucleotide Microarrays. Team This activity would be more beneficial by working with 1 to 2 other participants. Working as a team will promote discussion and further exploration into DNA microarray fabrication, operation and applications. Workspace A workspace with a flat table and plenty of elbow room is all you need to complete this activity. Supplies / Equipment SCME Kit – DNA Microarray Model Optional: A flashlight to simulate the UV light The kit contains  a “substrate” (below left) representing an 8 x 8 microarray (64 addresses / features),  many nucleotide bases (beads indicating A, C, G, or T),  a set of masks needed to fabricate a three nucleotide chain in each address, and  two sample bags – Sample 1 and Sample 2. (not shown below)

(Pictures may not match the new kit parts) Documentation 1. A table indicating the specific oligo for each feature in the area, 2. The cDNA sequence from the control or test samples that have attached to an oligo (indicate on the table). 3. Answers to the Post-Activity Questions. NOTE: Be sure to include ALL sources and references to data and graphics when applicable.



Activity: Building a DNA Microarray – Part I Procedure: Building the synthetic oligos 1. Check your supplies. You should have an 8 x 8 array, 4 different colors of beads, 12 masks, and a small bag of “fluorescent labeled nucleotide sequences” (3 beads and a sequin on a paperclip). 2. Note the legend at the top of the array. There are four nucleotide bases; therefore, there are four colors of beads, each color representing one specific nucleotide. Indicate the color for each nucleotide base. (Reminder: A nitrogenous base with a sugar and at least one phosphate is a nucleotide or nucleotide base.) a. Adenine - _______________ b. Thymine - _______________ c. Guanine - _______________ d. Cytosine -

_______________

3. Assuming the substrate (8 x 8 array) has been fully protected with a blocking agent, use Mask 1 to identify the features that will be deprotected with UV light. (Align Mask 1 to the array using the two pegs at the top of the board.) 4. At this time in a real fabrication process, UV light would be used to expose or deprotect the open features in the mask. If you have a flashlight, you can simulate this by holding the flashlight directly over the mask, turning the light ON for 2 seconds, then OFF.This step is deprotect. 5. “Add” the first nucleotide to the deprotected areas by dropping the correct color of bead through the holes. (The nucleotide base for each mask is indicated by both the color of the mask and the text at the top of the mask.) This step is addition. 6. Remove Mask 1. In a real fabrication process, the new nucleotides are protected with a blocking agent in preparation for the next mask. This step is protect. 7. Align Mask 2 to the array. Deprotect the select features. 8. “Add” the second nucleotide to the deprotected areas by dropping the correct color bead through the holes. 9. Continue to build your oligos using remaining masks. After Mask 12 you should have a 3 nucleotide oligo on each feature of your array. 10. Using the 8 x 8 array template (GeneChip® Model Template) on the next page, identify the oligo in each feature of the array. (e.g., TTT, TCA, CAT, AGG) 11. Complete the Part I: Post-Activity Questions.



DNA Microarray Model Template

1

2

3

4

5

6

7

8

A

B

C

D

E

F

G

H



Part I: Post-Activity Questions 1. Explain the function of the synthetic oligonucleotides on a DNA microarray.

2. How does a DNA microarray identify thousands of different genes (target ssDNA) simultaneously?

3. What is the purpose of the blocking agent in this fabrication process?

4. What is the purpose of the mask in each cycle of the fabrication process?

5. What happens during the “addition” step of the fabrication process?

6. Approximately how many masks would be required to fabricate a 30 base oligonucleotide in each feature of an 8 x 8 array?

7. Graphically, outline the fabrication process that utilizes photolithography and chemical reactions to build synthetic oligos. For each step, write a short description of the process.

An Online Review of DNA Microarray and Genechip® Fabrication Methods. Go to http://www.dnai.org/d/index.html Go go to “Genes and Medicine” then “Genetic Profiling”, then “Techniques”. Watch the sections for DNA Microarrays, GeneChips and then “Making GeneChips”



Interpreting Your Results Each feature (also called address) of a DNA microarray has a different set of synthetic oligo probes attached to it. Each feature may contain hundreds or thousands of identical probes, while each array may contain tens of thousands of features. Each feature is looking for a specific gene sequence (allele) found in a particular organism. Because an array contains thousands of features, it can simultaneously search and “find” thousands of specific genes. One way to help to ensure the accuracy of any DNA microarray test as well as to allow easier interpretation of the results, a control sample is used along with the test sample. Each gene in the control sample is tagged with a green fluorescent dye molecule. Each gene or cDNA in the target sample is tagged with a red fluorescent dye molecule. After hybridization, a laser scans the microarray and the presence of an attached target DNA is detected by the fluorescence of the label on the microarray. The image to the right is a scanned DNA microarray showing the hybridization results at each feature in the array.  Yellow indicates hybridization with cDNA from both control and test samples  Red indicates hybridization with cDNA from mostly or only the test sample  Green indicates hybridization with cDNA from mostly or only the control sample  Black indicates no hybridization with either sample Another way to ensure accurate test results is to build controls into the microarray during fabrication. Such control includes positive and negative control as well as direct comparison controls. Positive and Negative controls Positive and negative controls exist in all biological assays (tests that analyze biomolecules in specific events or conditions). The function of positive and negative controls is even more important for DNA microarrays because of the complexity of microarray fabrication. The purpose of positive and negative controls is to verify the overall performance of the microarray (Is it accurate and can it be trusted?) and the analytical technique (Were the samples prepared properly and the procedures executed correctly?). A negative control is an array feature that is designed to have NO binding or hybridization. Its purpose is to avoid getting “false-positives” – positive results that should have been negative. In the design of a DNA microarray, bogus features that have no oligos attached are interspersed throughout the array. If something does bind to one of these features, then the results of the test should be questioned and the test repeated. Prior to repeating the test, adjustments should be made to the preparation to better ensure accurate results. A positive control is one that you expect to show a positive result. An example is a feature that contains a gene sequence that is ALWAYS present; therefore, you should see hybridization with both the control and test DNA samples. If this positive control does not show hybrization with both samples, then one would have to assume that there may be “false negatives” elsewhere in Southwest Center for Microsystems Education (SCME) App_BioMEM_AC24c_PG_101014


the array. Therefore, the assay results should not be trusted. In the case of a “negative” positive control, one should examine the procedure used to prepare the DNA samples to ensure that it was correct. If the procedure was correct, then one should suspect that the microarray is defective. In this case, the test should be discarded. Direct Comparison Controls In this type of control, each feature of the array is a comparison of the test sample DNA to the control sample DNA. Such controls are for the direct comparison of each address (or each gene) between two different cells. The control DNA sample provides a comparison for evaluating the test DNA sample and thereby rules out other factors that might play a role in a low signal strength (low amount of test DNA binding or hybridization) or a high signal strength (high amount of test DNA hybridization). For example, if during fabrication of the microarray a more abundant density of capture oligonucleotide(probe) is made in one of the features, this could result in a higher amount of test DNA bound to that address in the array. Conversely, an address with a lower density of oligo synthesized will result in less test DNA bound there. By comparing a ratio of test DNA to control DNA bound at all of the arrays, it is possible to rule out these types of problems that can arise during fabrication.



Activity: Testing and Interpretation – Part II Note to the Instructor: The kit contains two bags of targets. Each bag contains targets from both the control sample and test sample. The targets from the control are the same in each bag; however, the test sample targets are different. Therefore, each bag yields different “results”. A chart showing the specific targets in each bag is provided on page 22. Mapping your array and identifying specific genes in the control and test samples NOTE: The purpose of Part II of this activity is to provide your with a little exposure into the world of DNA microarray interpretation. There is a tremendous amount of information supplied in a completed DNA microarray test. Therefore, one must have a strong foundation in biology and genetics to be able to understand and interpret the information that is available from such tests. This is only an exercise, not an actual test result. Procedure: 1. Ask your instructor for a bag of targets. Each bag of targets contain cDNA from both a control cell (sample) and a test cell. (Reminder: The cDNA in the control and test samples are specific gene sequences or alleles to be identified by the DNA microarray.) 2. Separate the target DNA sequences into control sample (green tags) and test sample (red tags). 3. Match each gene sequence (cDNA) to the complementary oligo on the array. Remember that the tag is at the TOP of the cDNA. 4. To your array template, indicate the features with a cDNA in the control sample and in the test sample by writing in the cDNA sequence and the specific sample. (For example -the feature for GGA would require a cDNA of CCT; therefore, if that match is made on your array with a green tagged sequence, your template would read “GGA-CCT (Gr)”.) [Review of DNA base pairs: A-T, T-A, G-C, C-G] Green

A G G

T C C

5. Answer the Post-Activity questions for Part II. Note to instructor: A microarray can be designed to identify all 30,000 genes of the human genome, as well as hundreds of thousands of mutant alleles of mutant genes. This microarray deals with 64 alleles and control features. A complete table of each feature is provided at the end of this Instructor Guide. The purpose of interpreting the results in this activity is to allow the participants to get an “idea” how the probes are used to identify a target or cDNA and create a hybrid and how specific features are used as test controls. Anything else is above and beyond the scope of this activity. Southwest Center for Microsystems Education (SCME) App_BioMEM_AC24c_PG_101014


Part II: Post-Activity Questions 1. The array features H5 and H7 are positive controls. a. What is the purpose of a positive control feature?

b. What can one infer when both positive control features have hybrids from both the control and test samples?

c. What would one see in the case of a “negative-positive” control?

2. The array features H6 and H8 are negative controls. a. What can one infer if either H6 or H8 or both indicate binding with a cDNA from either the control or test sample?

b. What can one infer if neither H6 nor H8 indicates binding with any of the cDNA from the samples?

3. What process is performed prior to testing to help distinguish between the targets in the control and test samples?

Following are tables that will assist you in the interpretation of this specific Genechip® test. a. The first table (List of the Gene Sequences in the Samples and the related Array Addresses) shows all of cDNA that are available in the control and test samples. The table briefly describes what gene sequence or allele is being “identified” in a specific array addresses. b. The second table (Notes for Interpretation of DNA Microarray) provides information on what a hybrid in a specific address may indicate.



Using the tables below, answer the following. Keep in mind that this is only an exercise to give you a taste of DNA microarray interpretation. There is A LOT more to it than is indicated here. 4. Is you test valid? _____________ a. If YES, explain.

b. If NO, explain.

5. Based on the results of your test, make assumptions as to the patient’s health and prognosis. Support your assumptions with specific results from the microarray. (If you have an invalid test, go ahead and complete an analysis of your results, assuming a valid test.)

List of the Gene in the Samples and the related Array Addresses Array Gene Name Address A5 A6 A7

BRCA1 BRACA1-11 BRCA2 BRCA2-26

Abbreviated Explanation Tumor suppressor gene that protects cells from cancer (normal allele) Mutation of BRCA1 tumor suppressor gene. This mutation is responsible for more than 80% of inherited breast and ovarian cancers Tumor suppressor gene that protects cells from cancer (normal allele)

B1

DAB

Mutation of BRCA2 tumor suppressor gene. This mutation is responsible for more than 80% of inherited breast and ovarian cancers Tumor suppressor gene (normal allele)

B2

DAB2

Mutation of DAB that enhances cell spreading

B7

ESR

Estrogen receptor cell signaling protein: proto-oncogene

B8

ESR1

Oncogene associated with breast and endothelial cancer.

C3

DCC

Tumor suppressor gene (normal allele)

DCC-02

A8

C8

IL6-01

One of 3 mutations of the DCC gene, associated with advanced colon cancer Oncogene associated with numerous cancers

D3

ID4

Tumor suppressor gene (normal allele)

F7

TP53

Tumor suppressor (normal allele)

TP53-D4

Mutation associated with smoking and confers an aggressive cancer with poor prognosis

C4

F8



H5

CP450

Positive Control (All cells have this gene) Negative Control

H6 H7

TUB

Positive Control (All cells have this gene)

H8

No oligo

Negative Control

Notes for Interpretation of DNA Microarray Array Address H5, H7 H6 and H8 B7 B8, C8

A5, A7, B1, C3, D3, F7

A6, A8, B2, C4, F8

Meaning of Green and/or Red A green and red on H5 and H7, AND H6 and H8 blank, validates the results. (It implies a properly fabricated array and the use of proper experimental procedures for making the oligos from the sample tissue.) Negative control. Both should be blank. A red or green marker in either of these addresses indicates an invalid test. A proto-oncogene (normal allele) Oncogene – Biologically, it only takes one oncogene allele to make cancerous tissue. This is because an oncogene is a dominant gene. Therefore, a red on this feature could indicate bad news for the patient. Tumor suppressors – Protect the cell from becoming cancerous. If one of the pairs of tumor suppressor alleles (e.g., BRCA1 and BRCA2) is mutated and no longer functions, the remaining tumor suppressor can compensate (i.e., BRCA1 can protect against a mutation of BRCA2). However, if both tumor suppressor genes are mutated (e.g., BRACA1-11 and BRCA2-26) and there is no normal allele left (e.g., BRCA1 or BRCA2), this is bad news for the patient. Gene mutations that aid in the development of cancer and may be used to determine the patient’s prognosis. If a feature for a normal allele is GREEN only, this means that only the mutant allele is found in the test sample DNA. If a feature for a mutant allele is BLACK, this means that neither the control nor the test sample has that mutation. If a feature for a mutant allele is RED, this means that the test sample has this mutation.



Abbreviated Glossary Allele – One member of a pair or variation of a gene that occupy a specific position on a specific chromosome. Gene–The basic biological unit of heredity. A length of DNA sequence or segment of DNA that “contributes to phenotype/function”.(Guidelines for Human Gene Nomenclature)This region is usually associated with regulatory regions of DNA sequence. Genome – An organism’s genetic material which is made up of molecules of DNA and found in the nucleus of eukaryotic cells and in the cytoplasm of prokaryotic cells. Hybridization–A process of combining two complementary single-stranded DNA into a single doublestranded molecule through base pairing. Nucleotides – Nitrogenous bases with a sugar and at least one phosphate, that when joined together, make up the structural unit of RNA and DNA. Oligonucleotide – A short fragment of single-stranded DNA typically 5 to 50 nucleotides long. Oncogene – A mutated gene that causes the transformation of normal healthy cells, into cancer cells. Proto-oncogene – A normal gene which, when altered by mutation, becomes an oncogene that can contribute to caner. Tumor Suppressor gene - A gene that protects a cell from cancer. If both pairs of tumor suppressor genes are mutated, the cell loses its protective effect and can become cancerous.



BONUS Activity: Designing a DNA Microarray Team Member Names: __________________ __________________

Objective: Design and simulate the fabrication of a DNA microarray by determining the DNA sequences for each of the features in a 4 x 4 array based a specific scenario. Once the sequences are determined, design the process masks that will yield those sequences.

Activity Part I: Design a 4 x 4 Direct Detection DNA microarray. Your array should meet the following criteria. (Use the notes and Activity Gene Table provided at the end of this activity to gather the information for the criteria.) 1. Design feature oligos to detect specific genes (3 nucleotides sequences) that could indicate a possible disease or trait (e.g., breast cancer, mild or aggressive) in a test sample. Create a scenario for your test. Write your scenario below. (e.g., This DNA test is to determine if the subject has the genes for breast cancer and if genes are present that indicate that the cancer may be of an aggressive or non-aggressive form.) _______________________________________________________________________________ _______________________________________________________________________________ _______________________________________________________________________________ 2. Each oligo sequence should be 3 nucleotides long and each feature should have one sequence. (Use the sequences and table at the end of this worksheet for a guide.) 3. Develop at least 12 unique sequences. (Identify sequences that would be found in both the test and control samples and at least two sequences found only in the test sample.) 4. Use controls to ensure test validity. 5. Complete the table on the next page to describe the content of each feature (the sequence, gene name, and whether the feature is a control, control gene, test gene or both).

DNA Microarray Layout In each of the array features, indicate… 1. the 3 sequence oligo (ATT) for that particular feature (first base at substrate) 2. the Gene Name from table at the end of this worksheet (e.g., BRCA1) Southwest Center for Microsystems Education (SCME) App_BioMEM_AC24c_PG_101014


3. whether it is a control, control gene, test gene or both

1

2

3

4

A

B

C

D



Activity Part II: Design the masks for your array. 1. Determine the sequence of nucleotide application for your microarray. (i.e., which nucleotide is applied first, second, third, etc.) 2. For your masks, you can either physically make the masks using paper or create a graphic or series of graphics that show the layout of each mask. Design the layout of each mask for each step. 3. Based on your sequences, how many masks did you need? ___________ 4. To test your masks, give them to another team and have it create an array from your masks

Post Activity Questions 1. What are the TARGET sequences that would hybridize with select features in your array yielding a “valid” test and showing the presence of at least one gene from the test sample only.

2. A DNA microarray can have as many as 30 bases in one oligo. What is the maximum number of masks that could be needed for an array with 30 base oligos?

3. Be prepared to state the purpose of each feature in your array.



Activity Gene Table (The DNA sequences in the features for the following gene are hypothetical and strictly for the purpose of this activity.) Array Address

Gene Name

Cellular Effects

A1

CDKN2

Tumor suppressor gene that encodes a protein called p16 that protects cells from cancer by stabilizing the tumor suppressor protein p23.

A2

One of 27 different mutations to CDKN2

A3

CDKN2A032 CDKN1

A4

CDKN1A-02

A5

BRCA1

Tumor suppressor gene that encodes a protein that plays a role in DNA repair and protects cells from cancer.

A6

BRCA1-11

A7

BRCA2

One of 20 mutations to BRCA1. Mutations of the BRCA tumor suppressor gene are responsible for more than 80% of inherited breast and ovarian cancers. Tumor suppressor gene that encodes a protein that plays a role in DNA repair and protects cells from cancer.

A8

BRCA2-26

B1

DAB

One of 20 mutations to BRCA1. Mutations of the BRCA tumor suppressor gene are responsible for more than 80% of inherited breast and ovarian cancers. Tumor suppressor

B2

DAB2

Mutation of DAB that enhances cell spreading.

B3

DRD

Proto-oncogene, dopamine receptor.

B4

DRD2

Oncogene that is associated with metastasis and confers a poor prognosis in cancer patients.

B5

END

Endothelin signaling protein: proto-oncogene.

B6

END1

Oncogene that is associated with aggressive cancer and confers a poor prognosis in cancer patients.

B7

ESR

Estrogen receptor cell signaling protein: proto-oncogene.

B8

ESR1

Oncogene that is associated with breast and endothelial cancer.

C1

ERBB2

The first proto-oncogene that was found, it stabilizes a family of growth factor cell receptors that are involved in triggering normal cell division.

C2

ERBB2-01

Mutation of ERBB2 that forms an oncogene that is linked to a poor prognosis in breast and ovarian cancer. This is one of 2 mutations of ERBB2

C3

DCC

Tumor suppressor

C4

DCC-02

One of 3 mutations that inactivate the tumor suppressor activity of the DCC gene, associated with advanced colon cancer.

C5

FHIT

C6

FHIT-01

Tumor suppressor gene that plays an important role in protecting the cell from carcinogens and in inducing cell death in response to DNA damage beyond repair. Mutations in this tumor suppressor gene have been found in about half of all esophageal, stomach, and colon carcinomas, and is a component of aggressive pancreatic cancer.

C7

IL6

Tumor suppressor encodes a protein called p21 that protects against cancer by inhibiting the action of cell signaling proteins and triggering cell death when DNA has been damaged beyond repair. Vitamin D activates the activity of p21, and this protein works with p53. One of 5 different mutations to CDKN1

This proto-oncogene is also known as: Interferon Beta-2 (IFNB-2), B-Cell differentiation factor, B-Cell stimulatory facto 2 (BSF2), Hepatocyte stimulatory factor (HSF) and Hybridoma growth factor (HGF).



C8

IL6-01

Oncogene associated with numerous cancers, especially lymphomas.

D1

Interferon regulatory factor. Deletions of this tumor suppressor are found in a number of leukemia’s.

D3

IFR1 (upstream sequence) IFR1 (downstream sequence) ID4

D4

ID4-01

Loss of the ID4 function results in aberrant methylation of DNA and is associated with leukemias and other cancers.

D5

INHA

Proto-oncogene that inhibits expression of certain growth factors.

D6

INHA

Oncogene often associated with prostate cancer.

D7

MYC

Proto-oncogene that encodes a protein that acts in the signaling pathway for normal cell proliferation.

D8

MYC-25

Leukemias and lymphomas are associated with 15 different mutations that create oncogenes of MYC.

E1

RB1

Tumor suppressor gene that encodes a protein that is a negative regulator of the cell cycle and was the first tumor suppressor gene found.

E2

RB1-01

This mutational defect of the RB1 tumor suppressor gene is a cause of childhood cancer retinoblastomas, bladder cancer, and osteogenic sarcoma.

E3

RNASEL

This tumor suppressor gene encodes a protein that helps to control cell division.

E4

RNASEL-12

One of 12 different mutations that have been linked to prostate cancer.

E5

SMAD4

Tumor suppressor gene.

E6

SMAD4-04

One of 3 mutations that inactive SMAD4, associated with advanced colon cancer.

E7

STAT3

Proto-oncogene.

E8

STAT3-03

One of 6 mutations of STAT3 that create an oncogene associated with ovarian, pancreatic, lung, renal, esophageal, cervical, colon, and gastrointestinal tumors.

F1

SS18

X-linked tumor suppressor

F2

SS18-01

Chromosomal translocation-associated oncogene associated with synovial sarcoma.

F3

ERG

Proto-oncogene

F4

TRPSS2

Chromosomal translocation of ERG and ETV1 creates an oncogene associated prostate, pancreatic, liver, lung and small intestinal cancers.

F5

TGFBR2

Tumor suppressor associated with RB1 function.

F6

TGFBR2-01

Inactivation of TGFBR2 associated with esophageal carcinoma.

F7

TP53

Tumor suppressor encodes a protein called p53 that protects against cancer by allowing DNA repair or triggers apoptosis when DNA damage is extensive.

F8

TP53-04

G1

PALB2

Inactivation of TP53 is associated with over 60% of all types of cancers. Over 19 mutations of TP53 have been characterized. This particular mutation is associated with smoking and confers an aggressive cancer and very poor prognosis. Tumor suppressor gene that promotes stabilized associations of the BRCA2 protein with DNA to allow for DNA repair.

D2

Interferon regulatory factor. Deletions of this tumor suppressor are found in a number of leukemia’s. Tumor suppressor gene that encodes Inhibitor of DNA Binding protein.



G2

FANCN

Mutation of PALB2 that is associated with cancers of the esophagus, breast, prostate, and stomach.

G3

XPA

Tumor suppressor gene that encodes a DNA excision enzyme that repairs DNA damaged by ultraviolet light (UV).

G4

XPA-01

Mutation of XPA is associated with skin cancer. Loss of function of XPA leads to aggressive cancer, with a poor prognosis.

G5

VHL

G6

VHL-01

Tumor suppressor gene encodes a protein involved in the ubiquitination and degradation of a transcription factor that plays a central role in the regulation of gene expression by oxygen. Loss of function of VHL leads to cancers of the cerebellum, spinal cord, kidney and eye.

G7

WRN

Tumor suppressor gene that encodes a helicase involved in DNA repair.

G8

WRN-01

Loss of function of WRN leads to defective DNA repair and aggressive forms of cancer.

H1

ACD

H2

ACD-01

A guardian gene. This gene encodes telomerase. In differentiated cells, this enzyme is not expressed, limiting the number of cell divisions that they can undergo. A telomerase that is inappropriately expressed in cancer cells allow them to divide indefinitely.

H3

GAPC

Glyceraldehyde phosphate dehydrogenase, a glycolytic enzyme essential to all living organisms. A “housekeeping” gene.

H4

H1

All cells have histone genes that provide the structural basis of nucleosomes in the chromatin. A “housekeeping” gene.

H5

CP450

All cells must have functional cytochrome p450 genes. A “housekeeping” gene.

H6

No oligo

(negative control reaction)

H7

TUB

All cells have functional tubulin genes that provide cytoskeletal microtubules. A “housekeeping” gene.

H8

No oligo

(negative control reaction)



Summary DNA microarray fabrication requires the construction of thousands or millions of oligonucleotides within the features of an array. One fabrication process uses the technology of the inkjet printer while another process uses photolithography. The photolithography process results in synthetic oligos built one nucleotide at a time using specially designed masks and UV light. Each of these processes has the ability to construct large microarrays capable of identifying thousands of different genes simultaneously. References 1. Antler, B. C. (2004, August). Spot Your Genes - an Overview of the Microarray. Retrieved from The Science Creative Quarterly: http://www.scq.ubc.ca/spot-your-genes-an-overviewof-the-microarray/ 2. Stimson, D. (2007, July 29). After a decades-long search, scientists identify new genetic risks factors for multiple sclerosis. Retrieved from EurekAlert!: http://www.eurekalert.org/pub_releases/2007-07/nion-aad072607.php 3. Zaccheo, R. K. (2005). GeneChip Microarrays Curriculum for Life Science Teachers. Retrieved from IISME: http://www.iisme.org/etp/HS%20Science%20GeneChip%20Mciroarrays.pdf Disclaimer The information contained herein is considered to be true and accurate; however the Southwest Center for Microsystems Education (SCME) makes no guarantees concerning the authenticity of any statement. SCME accepts no liability for the content of this unit, or for the consequences of any actions taken on the basis of the information provided.

Support for this work was provided by the National Science Foundation's Advanced Technological Education (ATE) Program. This Learning Module was developed in conjunction with Bio-Link, a National Science Foundation Advanced Technological Education (ATE) Center for Biotechnology @ www.biolink.org.



DNA Microarray Activity: An Ethical Dilemma? Participant Guide Description and Estimated Time to Complete Thisactivity provides you the opportunity to evaluate your own ethical decisions in various situations and to discuss with others the ethical dilemma surrounding some of the applications of DNA microarrays. After completing this activity you should be able to describe and justify your personal opinions about the use of DNA microarray in certain applications and better understand the opinions of others in answering the question “Are DNA microarrays an ethical dilemma?” Estimated Time to Complete Allow at least 60 minutes to complete. Introduction What are ethics?You can think of ethics as being the study of why some specific actions may be right or wrong, praiseworthy or blameworthy. Being ethical is not just following the law or the rules that dictate society, but using your personal beliefs in forming your decisions. "Ethics refers to well based standards of right and wrong that prescribe what humans ought to do, usually in terms of rights, obligations, benefits to society, fairness, or specific virtues."1 However, one can always find arguments for and against the existence of the concepts of „right‟ or „wrong.” This is why ethics sometimes differs from one person to another, from one society to another, from one situation to another. Think of a personal experience where you disagreed with someone. Was the disagreement due to differences in your personal beliefs or values? How did you feel about having your beliefs challenged?

Southwest Center for Microsystems Education (SCME) App_BioMEM_AC24d_PG_102813

Page 1 of 6 DNA Microarray – An Ethical Dilemma? Activity

What is ethical?Ethical issues are complex. They vary with each situation. Ethical issues can arise anytime we consider an action as being morally right or wrong. Such actions can occur when humans, animals, living things or historical artifacts are being manipulated for the benefit of others. Some examples could be unfair employment practices, testing chemical side effects on animals, or tearing down a historical landmark to build a parking lot.In other words, would it be morally wrong for people to achieve personal gains without regard to others, such as selling a product that could hurt the buyer or a product that you know they don‟t need and can‟t afford? Ethical issues can be something as simple as where to park. For example, what choice would you make in the following situation? An ethical dilemma: On your way home from school or work, you stop off at the local grocery to get a couple of items. The parking lot is pretty full, but a handicap spot is open near the door. You have to make the choice of parking in the handicap spot (since you only have 2 items to get) or park in the next closest spot about 30 to 40 feet away. What choice would you make? Why do we need to discuss ethics? It is clear that many areas of life contain questions concerning right and wrong. How people answer these questions is dependent on their personal ethics which are not always the same as yours. One‟s ethics are unique. How many other people would make the same choice as you did in the previous situation? People base ethical decisions on their personal values and principles. These values and principles are developed through individual life experiences and beliefs. Ethical principles are general statements of how people should or should not act in most situations. Principles are often the reasons behind our actions. Ethical principles need to be addressed in all areas including science and technological developments. In this lesson you will examine the ethical challenges of new technology, specifically the DNA microarray in reference to human health and safety.



Activity Objectives Activity Objectives  State how you would respond to a situation in which one might find unethical.  Justify your personal opinion on the ethics of using DNA microarray for certain applications. Resources Computer with high-speed Internet access. Documentation This activity has two parts. Your documentation should include all of the questions asked in each part of the activity and your answer to each of these questions. In Part II, your documentation should include a summary of your group‟s analysis of the situation and the group‟s discussion. Your summary should also include the opinions that were expressed on the ethics of the situation and the personal ethics of group members in forming their opinions and final decision on what should or should not be done in this situation.

Activity – Part 1: What is ethical? PROCEDURE – Part I: 1. Analyze each of the following scenarios. For each scenario answer the questions: a. What is the ethical situation presented (if there is one)? b. What is "right" or "wrong"? c. What do you believe is the cause of the problem (if there is a problem)? d. What are some ideas for possible solutions to the problem? e. Which of these solutions do you feel is the best? Why? f. Who would benefit from your decision? g. Who would lose due to your decision? Scenario 1: In a parking lot, you open your car door too forcefully and leave a big scratch as well as a dent on the car next to yours. What would you do? Scenario 2: You witness a hit and run along with 5 of your friends. There are several other people in the area that have also witnessed the hit and run. Your friends start walking away from the scene, heading toward your original destination. What would you do?



Activity – Part II: DNA Microarrays – An Ethical Dilemma? There have been many questions raised on the ethics of DNA microarrays for certain applications and possible uses or abuses. Two questions we all need to ask are 

“Do these applications hurt society more than help it?



Are these applications ethically "right" or "wrong"?

Every time the human race makes a new discovery or introduces new technology, the question "Is it ethical?" is asked. Reaching a unanimous decision is impossible because people's ethics differ; however, there are some common rights and wrongs on which people can reach consensus. In this part of the activity you will research a specific application of DNA microarrays and then have a discussion with other participants on the ethics of using DNA microarrays for that application. Before beginning your discussion make sure that you understand the DNA microarray application and the ethical concerns surrounding it. Let‟s review some of the rules that should be applied when discussing controversial issues. Rules to Apply  Identify the ethical question or situation.  Determine if the information given is reliable.  Ask: "Is there something that is not been said?"  If you feel like something is missing, then research the situation, learn more about it and get a better understanding of it.  Determine who is responsible for what.  Identify who may or may not be affected.  Based on your analysis and facts, determine what should be done and by whom.  Be able to justify your final decision as to what should be done.



PROCEDURE – Part II 1. Read through the following scenarios that present “questionable” applications of DNA microarrays. With your group, decide which scenario you would like to discuss. If you are unfamiliar with the application or you need more information, your group may decide to do some additional research before discussing it so that you can better support your opinions. Scenario 1: UC Berkeley Plan to Test Freshmen DNA Criticized Marcus Wohlsen, Associated Press Writer Drug Discovery & Development - May 21, 2010

http://www.dddmag.com/news-UC-Berkeley-Plan-to-Test-Freshmen-DNA-Criticized.aspx Summary:“A plan by the University of California, Berkeley to voluntarily test the DNA of incoming freshman has come under fire from critics who said the school was pushing an unproven technology on impressionable students. The university has said it will send test kits to 5,500 new students to analyze genes that help control the body's responses to alcohol, dairy products and folic acid.” Scenario 2: Prenatal DNA test raises both hopes and worries. Roger Collier. Canadian Medical Association Journal (CMAJ) 2009 March 31; 180(7): 705-706. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2659835/ Summary:“A new prenatal genetic diagnostic test may soon cause a substantial increase in the number of fetuses affixed with “syndrome” labels. The noninvasive test, called chromosomal microarray analysis, allows doctors to detect submicroscopic genetic abnormalities that no other test can find. Advocates of the technology say it is safer, faster and more accurate than invasive diagnostic procedures like amniocentesis. Despite the test's benefits, however, some worry that it will result in a flood of prenatal genetic information of uncertain significance and will lead only to confusion and undue anxiety for expectant parents. Others question whether scientists should even be in the business of cleaning up the gene pool and have evoked the dreaded “E” word: eugenics.” Scenario 3:

Identifying Economically Important Traits in Animal Genomes.Stacy Kish, CSREES Staff(2009, November 27). National Institute of Food and Agriculture. http://www.nifa.usda.gov/newsroom/impact/2009/nri/01281_animal_genomes.html Summary:SNP (single nucleotide polymorphisms) chips, a type of direct detection DNA microarray, have been developed that “easily and quickly identify regions within the bovine genome that harbor variants that cause the animals to differ in the outward expression of important traits, allowing scientists to predict an animal’s genetic merit from its SNP profile.” The results of these analyses can be used to “to guide the

genetic selection process of milk-producing animals.”



2. As a group, discuss the ethical dilemma that may or may not be associated with one of these scenarios. Use the following rules as a guide for your discussion.  Identify the ethical question or situation.  Determine if the information given is reliable.  Ask: "Is there something that is not been said?"  If you feel like something is missing, then research the situation, learn more about it and get a better understanding of it.  Determine who is responsible for what.  Identify who may or may not be affected.  Based on your analysis and facts, determine what should be done and by whom.  Be able to justify your final decision as to what should be done. 3.

Document your group‟s analysis of the situation and summarize your discussion. Your summary should include the opinions that were expressed, the ethics of the situation and the personal ethics of group members in forming their opinions and final decision on what should or should not be done in this situation.

Summary In this activity you learned a methodical and objective way to analyze potentially ethical situations.Using this method, you should have been able to evaluate your own ethics in certain situations and to be able to discuss difficult issues in a non-threatening and productive manner. References 1

What is Ethics? Developed by Manuel Velasquez, Claire Andre, Thomas Shanks, S.J., and Michael J. Meyer Markkula Center for Applied Ethics, Santa Clara University. http://www.scu.edu/ethics/practicing/decision/whatisethics.html

2

“Ethics Module , Ambient Program, NIEHS http://www.rsmas.miami.edu/groups/niehs/ambient/ To contact Ambient:[email protected]

3

The Ethics of NanoTechnology – NanoTechnology Now, February 7, 2007: http://www.nanotech-now.com/ethics-of-nanotechnology.htm

4

Responsible Nanotechnology: Looking Beyond the Good News, Vicki Colvin, Director of the Center for Biological and Environmental Nanotechnology, Rice University: EurekAlert! http://www.eurekalert.org/context.php?context=nano&show=essays&essaydate=1102




Southwest Center for Microsystems Education (SCME) Learning Modules available for download @ scme-nm.org MEMS Introductory Topics

MEMS Fabrication

MEMS History MEMS: Making Micro Machines DVD and LM (Kit) Units of Weights and Measures A Comparison of Scale: Macro, Micro, and Nano Introduction to Transducers Introduction to Sensors Introduction to Actuators Problem Solving – A Systematic Approach Wheatstone Bridge (Pressure Sensor Model Kit)

Crystallography for Microsystems (Crystallography Kit) Deposition Overview Microsystems Photolithography Overview for Microsystems Etch Overview for Microsystems (Rainbow Wafer and Anisotropic Etch Kits) MEMS Micromachining Overview LIGA Micromachining Simulation Activities (LIGA Simulation Kit) Manufacturing Technology Training Center Pressure Sensor Process (Three Activity Kits) MEMS Innovators Activity (Activity Kit) A Systematic Approach to Problem Solving Introduction to Statistical Process Control

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