Microarray Technology

Asma-ul-husna07-arid-1237

Ph.D (Zoology)1st Semester

Microarray

Microarray is a tool for analyzing gene expression that consists of a small membrane or glass slide

containing samples of many genes arranged in a regular pattern.”

History• Microarray technology evolved from Southern

blotting, where fragmented DNA is attached to a substrate and then probed with a known DNA sequence.

• The use of miniaturized microarrays for gene expression profiling was first reported in 1995, and a complete eukaryotic genome (Saccharomyces cerevisiae) on a microarray was published in 1997.

Introduction

• The underlying principle of microarray technology is the ability of DNA to bind to itself and to RNA.

• Analyzing gene expression involves the detection of mRNA species (transcriptome) present in a cell or tissue at a particular point in time.

Principle• DNA microarrays have provided a new and powerful tool to

perform important molecular biology and clinical diagnostic assays.

• The basic idea behind DNA microarray technology has been to immobilize known DNA sequences referred to as probes in micrometer-sized spots on a solid surface (microarray) and specifically hybridize a complementary sequence of the analyte DNA or a target.

• A fluorescently labeled reporter facilitates fluorescent detection of the presence or absence of a particular target or gene in the sample.

• By using laser-scanning and fluorescence detection devices such as CCD cameras, different target hybridization patterns can be read on the microarray and the results quantitatively analyzed.

The plate

• Usually made commercially.

• Made of glass, silicon, or nylon.

• Each plate contains thousands of spots, and each spot contains a probe for a different gene.

• A probe can be a cDNA fragment or a synthetic oligonucleotide, such as BAC (bacterial artificial chromosome set).

• Probes can either be attached by robotic means, where a needle applies the cDNA to the plate, or by a method similar to making silicon chips for computers. The latter is called a Gene Chip.

Procedure

1) Collect Samples.

2) Isolate mRNA.

3) Create Labelled DNA.

4) Hybridization.

5) Microarray Scanner.

6) Analyze Data

1: Collect Samples

• This can be from a variety of organisms. We’ll use two samples – cancerous human skin tissue & healthy human skin tissue

2: Isolate mRNA.

• Extract the RNA from the samples. Using either a column, or a solvent such as phenol-chloroform.

• After isolating the RNA, we need to isolate the mRNA from the rRNA and tRNA. mRNA has a poly-A tail, so we can use a column containing beads with poly-T tails to bind the mRNA.

• Rinse with buffer to release the mRNA from the beads. The buffer disrupts the pH, disrupting the hybrid bonds.

3: Create Labelled DNA Add a labelling mix to the RNA.

The labelling mix contains poly-T (oligo dT) primers, reverse transcriptase (to make cDNA), and fluorescently dyed nucleotides.

We will add cyanine 3 (fluoresces green) to the healthy cells and cyanine 5 (fluoresces red) to the cancerous cells.

The primer and RT bind to the mRNA first, then add the fluorescently dyed nucleotides, creating a complementary strand of DNA

4: Hybridization

• Apply the cDNA we have just created to a microarray plate.

• When comparing two samples, apply both samples to the same plate.

• The ssDNA will bind to the cDNA already present on the plate.

5: Microarray Scanner

The scanner has a laser, a computer, and a camera.

The laser causes the hybrid bonds to fluoresce.

The camera records the images produced when the laser scans the plate.

The computer allows us to immediately view our results and it also stores our data.

6: Analyze the Data GREEN – the healthy sample hybridized

more than the diseased sample.

RED – the diseased/cancerous sample hybridized more than the nondiseased sample.

YELLOW - both samples hybridized equally to the target DNA.

BLACK - areas where neither sample hybridized to the target DNA.

By comparing the differences in gene expression between the two samples, we can understand more about the genomics of a disease.

6. Continued Biological Samples in 2D Arrays on Membranes or Glass Slides

Whole process through animation

• http://highered.mcgrawhill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120078/micro50.swf::Microarray

• http://www.digizyme.com/competition/examples/genechip.html

Types of microarrays

It include:

• DNA microarrays,

• oligonucleotide microarrays

• Protein microarrays

• Tissue microarrays

• Cellular microarrays (also called transfection microarrays)

• Chemical compound microarrays

• Antibody microarrays

• Carbohydrate arrays (glycoarrays)

Design of a DNA microarrays

The property of complementary nucleic acid sequences is to specifically pair with each other by forming hydrogen bonds between complementary nucleotide base pairs.

The principle of DNA microarrays lies on the hybridization between the nucleotide. Using this technology the presence of one genomic or cDNA sequence in 1,00,000 or more sequences can be screened in a single hybridization.

Oligonucleotide Microarray

Protein Microarray

Tissue & Antibody Microarray

TRANSFECTION MICROARRAY (A) Outline of the procedure (B) An example of the transfection

microarray format. HEK 293 cell line was transfected.

Chemical compound microarray

Carbohydrate Microarray

Applications

The DNA chips are used in many areas as given below:

• Gene expression profiling

• Discovery of drugs

• Diagnostics and genetic engineering

• Alternative splicing detection

• Proteomics

• Functional genomics

• DNA sequencing

• Toxicological research (Toxicogenomics)

ADVANTAGES

• Provides data for thousands of genes.

• One experiment instead of many.

• Fast and easy to obtain results.

• Huge step closer to discovering cures for diseases and cancer.

• Different parts of DNA can be used to study gene expression.

Disadvantages

• The biggest disadvantage of DNA chips is that they are expensive to create.

• The production of too many results at a time requires long time for analysis, which is quite complex in nature.

• The DNA chips do not have very long shelf life, which proves to be another major disadvantage of the technology.

Summary• DNA Microarrays are one of the most effective invention ever

developed.

• A DNA Microarray is a test that allows for the comparison of thousands of genes at once.

• Microarray technology uses chips with attached DNA sequences as probes for gene expression.

• Any DNA in the sample that is complementary to a probe sequence will become bound to the chip.

• Microarray technology is most powerful when it used on species with a sequenced genome. The microarray chip can hold sequences from every gene in the entire genome and the expression of every gene can be studied simultaneously.

• Gene expression data can provide information on the function of previously uncharacterized genes.

Thank you for your patience

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