DNA Microarray introdution and application

DNA MICROARRAY

Neeraj Sharma MSc Medical Microbiology2nd year

What we will be discussing…

• What is DNA microarray?• The purpose of using DNA microarray.• Principle • Steps to perform a microarray.• Types• Applications

Introduction • DNA chip or a Biochip.• Collection of

microscopic DNA spots attached to solid surface.

• The probe sequences are designed and placed on an array in a regular pattern of spots.

• The chip or slide is usually made of glass or nylon and is manufactured using technologies developed for silicon computer chips.

• Each microarray chip is arranged as checkerboard of 105 or 106 spots or features, each spot containing millions of copies of a unique DNA probe (often 25 nt long).

• Each spot contains pico moles of (10-12 moles) of specific DNA sequence known as probe.

Purposes.• So why do we use DNA microarray?

o To measure changes in gene expression levels – two samples’ gene expression can be compared from different samples, such as from cells of different stages of mitosis.

o To observe genomic gains and losses. Microarray Comparative Genomic Hybridization (CGH)

o To observe mutations in DNA.

Principle • The core principle behind

microarrays is hybridization between two DNA strands, the property of complementary nucleic acid sequences to specifically pair with each other by forming hydrogen bonds between complementary nucleotide base pairs.

• A high number of complementary base pairs in a nucleotide sequence means tighter non-covalent bonding between the two strands. After washing off non-specific bonding sequences, only strongly paired strands will remain hybridized.

Steps1) Collect Samples.2) Isolate mRNA.3) Create Labelled DNA.4) Hybridization.5) Microarray Scanner.6) Analyze Data.

STEP 1: Collect Samples.

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

STEP 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.

STEP 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

STEP 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.

STEP 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.

STEP 6: Analyze the Data.

GREEN – the healthy sample hybridized more than the diseased sample.

RED – the diseased/cancerous sample hybridized more than the non-diseased 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.

Types• Spotted• DNA fragments (usually

created by PCR)or oligos are stuck to glass slides

• The size of the fragment can be any length (usually 500 bp-1 kb)

• The size of the oligos range from 20-100 nts

• These arrays can be created in individual labs using “affordable” equipment

• Affymetrix• Affymetrix arrays are

typically limited to oligos of 20-25 nts

• The probes on these arrays are synthesized using a light mask technology

• Photo-sensitive reactions are used to remove a blocking group and then extend

• It is very costly to fabricate masks for a new array design

• Not commonly used for custom arrays

Applications • Medical field : - Cause of disease Disease identification Disease treatment

• Agriculture

• Forensic

• Toxicology

• Detect the presence or absence of specific genes.

• Compare genes from two different sources.

• They can see how the external genes are affecting the external stimuli.

Printing of the probes in microarray.

Thank You