DNA MICROARRAYS. DNA Microarray

Transcription

1 DNA MICROARRAYS DNA Microarray Micro Nano Tech Conference I

2 DNA Microarrays Revised 05/05/11 DNA microarrays help us 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 researchers have been able to identify specific genes that influence the risk of getting a disease. 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. 2

3 Review of DNA Before jumping into the DNA microarray, let s review the Deoxyribonucleic acid (DNA) molecule, DNA transcription, and DNA hybridization. Please refer to the glossary at the end of the Primary Knowledge unit if you get stuck on terminology. 3 Revised 05/05/11

4 What is DNA? DNA is a long polymeric molecule (found in most cells) that functions in the chromosome as the carrier of genetic information. The genetic information is stored in the linear sequences of the base pairs (nucleotides): Adenine-Thymine (A-T or T-A) Guanine-Cytosine (G-C or C-G) One DNA molecule may consists of millions of nucleotides and thus, millions of linear sequences (genes). A, T, G, and C are the nitrogenous bases. When paired, they are called base pairs or nucleotides. Each base pair is joined with a weak hydrogen bond. 4

5 Your Genes An organism s genome is its hereditary information carried in DNA. The Human Genome project (1990 through 2003) mapped approximately 30,000 linear sequences or human genes. Every single cell in the human body contains the exact same genes; however, some are active and other s are not. DNA Microarrays identify specific genes as well as the activity of genes. (More on that later.) DNA microarrays use copies of one s DNA. 5

6 DNA Transcription DNA copies are made through DNA Transcription followed by reverse transcription. Transcription creates a messenger Ribonucleic acid molecule (mrna) from a DNA molecule. The DNA sequences (genes) from one half of the DNA molecule are copied into the mrna. 6

7 Reverse Transcription Through reverse transcription the mrna sequences are transferred into a DNA copy or cdna. DNA microarrays require the cdna rather than the mrna, because mrnas are unstable, thus leading to inaccurate and unreliable results. cdna are less likely to degrade during the hybridization process in a microarray. 7

8 DNA Hybridization DNA hybridization is when a single-stranded DNA molecule (ssdna or the cdna) reanneals with another ssdna from another source. If the cdna strands have sequences that are complementary to the introduced ssdna strands, they can form a dsdna hybrid molecule with one strand from each. 8

9 DNA Hybridization Animation Play QuickTime Animation 9

10 DNA Microarrays and Hybridization DNA microarrays use gene sequencing, DNA transcription and reverse transcription, and hybridization to analyze and identify thousands of genes simultaneously. Each microarray consists of hundreds or thousands of gene sequences (or ssdna molecules) mounted on a chip and used as probes. Theses probes are called oligonucleotides or oligos. 10

11 What is a DNA Microarray? The DNA microarray relies on the hybridization of cdna fragments to synthetic ssdna sequences (specific A, C, G, T combinations). The synthetic fragments (oligos) are the probes. The cdna fragments are the targets. 11

12 DNA Microarrays A DNA microarray is a grid on a substrate (e.g., glass, silicon). Each position in the grid is an address or feature as small as 200 nm square. Each feature may contain hundreds or thousands of identical probes (oligos). Each array may contain tens of thousands of features. Each feature is looking for a specific gene or gene sequence. Thousands of specific genes can be identified simultaneously. 12

13 DNA Microarrays This graphic illustrates one feature of a DNA microarray expanding 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 (oligos) in a DNA microarray. DNA, and thus a DNA microarray is actually colorless. In the middle graphic, how many gene sequences are being tested? 13

14 DNA Microarrays chips The image below shows a DNA microarray with tens of thousands of features (left) and an exploded section of the array (right). Each colored dot is one feature or address containing hundreds/thousands of the same oligo probe that have hybridized with cdna from a sample. [Courtesy of Affymetrix] We ll explain the colors in a later slide. 14

15 DNA Microarray Controls Controls are used to ensure the test results by ensuring the accuracy of the microarray s fabrication and the preparation of the samples (control and test samples). Control sample A set of cdna from tissue for which the genes are known. Test sample A set of cdna from tissue that is to be analyzed. Positive control A feature that must show hybridization with both the control sample and test sample. Negative control A feature that must show NO hybridization with any cdna from either sample. Direct Comparison controls Each feature of the array is a comparison between the control sample and the test sample (patient). 15

16 How Does a DNA Microarray Work? [Courtesy of "The Science Creative Quarterly". scq.ubc.ca. Artist: Jiang Long} DNA Microarrays use a control and target or experimental cell. 1. For each cell, mrna is extracted from the active DNA in the cells. 2. cdna is reversed transcribed from the mrna. 3. Each cdna is fluorescently labeled green (control) and red (target). 4. The samples are combined and washed over the array. 5. Complementary genes hybridization with the synthetic probes on the array. 16

17 Interpretation of Microarray Results Revised 05/05/11 [Image courtesy of NASA] A flourescening DNA microarray showing the results of DNA hybridization between the probes and target DNAs. When cdna is prepared from a test sample (red) and from a control sample (green), and both hybridize with probes, the color of the dot indicates the relative activity level or the presence of that gene in one or both samples. Green dot shows activity or presence in the control only Red shows activity or presence in the test tissue only Yellow shows activity/presence in both. Black is activity/presence with neither sample. 17

18 Types of DNA Microarrays Gene expression microarrays detect expression levels in a sample - when mrna copies to cdna (which genes are active or inactive ). 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. Direct detection microarrays are 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. 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 tissue from a patient and each column a gene. [This image is public domain] 18

19 DNA Microarray Animation Play QuickTime Animation 19

20 DNA Microarray Applications Identify specific genes that cause a specific disease Identify gene mutations Compare genes between different organisms Create gene expression profiles (A map that shows gene activity or the making of mrna. Gene expression profiles detect how cells and organism change and adapt to specific stimuli such as change s in environment or one s disease state.) Study cancer causing genes Customize drug therapies based on one s personal genes 20

21 DNA Microarray Fabrication So how on earth do we fabricate a DNA microarray? 21 Revised 05/05/11

22 GENECHIP MODEL ACTIVITY DNA Microarray Learning Module

23 Activity Description Revised 05/05/11 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. This activity should improve your understanding of how DNA microarrays are made and how they identify complementary single-stranded DNA (ssdna) in a sample. 23

24 Activity Objectives and Outcomes Using the components provided in a SCME DNA Microarray kit, build a macro-size DNA microarray with a three (3) nucleotide sequence. Outline and explain the fabrication steps for an oligonucleotide array or GeneChip. Revised 05/05/11 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? 24

25 DNA Microarray Fabrication 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 that deposits one nano-size nucleotide at a time, creating the desired nucleotide sequences (or oligonucleotides). 25

26 DNA Microarray Fabrication The inkjet 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 are synthesized by chemical methods, one at a time, or are prepared enzymatically by a reverse transcription of mrna isolated from cells to copy DNA or cdna. Alternatively, a photolithography method uses the photolithography process borrowed from the semiconductor fabrication industry in combination with chemical reactions to synthesize oligonucleotide (oligo) probes on a silicon surface. The oligos on these arrays are generally 20 to 25 nucleotides long and each feature in the array itself may be as small as 50 nm square almost 2000 times smaller than the width of a strand of hair! 26

27 Microarray Fabrication - Photolithography This method duplicates the photolithography process used in microfabrication along chemical reactions between silicon and a nucleotide, and between different nucleotides. A patterned mask and ultraviolet (UV) light expose specific nano-size features of the microarray. Each feature is a location for several nucleotides of the same sequence. The graphic illustrates the UV light, mask, and substrate. Each square in the mask is a select feature or address in the array. Inside each feature are several silicon molecules that will link with the initial nucleotides. This is the method that we will explore in the GeneChip Model Activity. This method was developed by Affymetrix and is used to fabricate synthetic oligonucleotides on a silicon substrate. 27

28 Fabrication of a Oligonucleotide Array The light that travels through these holes initiates the growth of a nucleotide chain through a process of deprotection and addition of new bases into specific chains / probes. The chain begins with a nucleotide linking to an unprotected silicon molecule on the surface of the substrate. The mask is therefore the tool that ultimately controls the building of the oligo probes. 28

29 Three Step Process Protect Initially, a blocking compound is washed over the entire silicon wafer. In subsequent steps, the blocking compound is already attached to the nucleotides in the Addition step. Deprotect (Photolithography) UV light through the holes in the mask, removes the blocking compound, deprotecting that area. Addition The substrate is washed with a solution of the specific nucleotide being added at that step. The nucleotides in the solution attach to the deprotected areas on the wafer. This cycle is repeated for each new nucleotide. 29

30 Genechip Animations Let s take a look at a couple of animations. GeneChip Animation on YouTube DNA Microarray by Affymetrix 30

31 Let s to do it! Are you ready? Let s build a three nucleotide sequence DNA microarray! 31

32 GeneChip Model Activity So let s step through activity materials and fabrication process together. The board simulates an array on a silicon substrate. (Yours may look a little different from the one in the picture.) By the end of this activity, each feature in the array will have an oligo of 3 nucleotides. 32

33 GeneChip Model Activity In this activity you will fabricate a 3 nucleotide oligo (nucleotide sequence) using 12 masks. Each mask identifies the locations for one of four nucleotides (A, C, T, or G) with a hole or opening. Colored beads are used for each nucleotide. Refer to the top of your substrate (board) for the color of bead vs. the specific nucleotide. 33

34 GeneChip Model Activity (Fabrication) 1. Set up your station with your substrate, beads (nucleotides), and mask (cards with the pretty flowered holes). 2. Using mask 1, align it over the substrate using the 2 alignment pegs at the top of the board. 3. Mask 1 is for the T nucleotides. Drop a T bead through each hole and onto a nail. (You have just exposed for and added a T nucleotide. 4. Remove the Mask 1. Align Mask 2 for the C nucleotide. 5. Add C to all of the deprotected areas. 6. Continue this process through all 12 masks. 7. On the array table in your activity, write the oligo for each feature. 34 Revised 05/05/11

35 Interpretation of Microarray Results A flourescening 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 activity in neither sample. [Image courtesy of NASA] Revised 05/05/11 35

36 GeneChip Model Activity (Interpretation) Revised 05/05/11 Remember this graphic? Ask your instructor for a sample bag. In your bag are fluorescently labeled nucleotides (red or green sequin with 3 beads). These are cdna targets from a control cell and test cell. Which is which? Match your targets with the oligo probes on your array. Which genes are detected in the control and in the target? 36

37 Summary Revised 05/05/11 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. 37

38 Incorporating biomems How could you incorporate the information from this workshop into your curriculum? 38 Revised 05/05/11

39 THANK YOU FOR COMING!!!! Please check the SCME website periodically - scme-nm.org. We are always uploading new materials and scheduling new workshops. Feel free to participant in our forum to present ideas, results, and ask questions. 39 Revised 05/05/11