DNA MICROARRAYS - scme-nm.org€¦ · 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 MICROARRAYS

Micro Nano Tech Conference I - 2011

DNA Microarray

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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.

DNA Microarrays

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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.

Review of DNA

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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.

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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.

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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.

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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.

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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.

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DNA Hybridization Animation

Play QuickTime Animation

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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.

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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.

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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.

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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?

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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.

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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).

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How Does a DNA Microarray Work?

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.[Courtesy of "The Science Creative Quarterly". scq.ubc.ca. Artist: Jiang Long}

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Interpretation of Microarray Results

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.

[Image courtesy of NASA]

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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]

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DNA Microarray Animation

Play QuickTime Animation

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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

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So how on earth do we fabricate a DNA

microarray?

DNA Microarray Fabrication

GENECHIP MODEL

ACTIVITY

DNA Microarray Learning Module

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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.

Activity Description

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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®.

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?

Activity Objectives and Outcomes

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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).

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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!

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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.

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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.

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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 deprotectedareas on the wafer.

This cycle is repeated for each new nucleotide.

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“Genechip” Animations

Let’s take a look at a couple of animations.

GeneChip Animation on YouTube

DNA Microarray by Affymetrix

http://www.youtube.com/watch?v=V8uNJCO7Qqo

http://www.youtube.com/watch?v=ui4BOtwJEXs&feature=related

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Let’s to do it!

Are you ready?

Let’s build a three nucleotide sequence DNA

microarray!

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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.

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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.

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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.

GeneChip Model Activity (Fabrication)

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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]

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GeneChip Model Activity (Interpretation)

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?

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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.

Summary

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How could you incorporate the information

from this workshop into your curriculum?

Incorporating bioMEMS

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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.

THANK YOU FOR COMING!!!!

Documents

DNA MICROARRAYS - scme-nm.org€¦ · a DNA microarray expanding from many features (bottom grid) to a few features (middle grid), to a single feature (top grid) depicting a unique