DNA MICROARRAY

NAVEED UL MUSHTAQDept. : BIO-RESOURCESUNIVERSITY OF KASHMIR

INTRODUCTIONDEFINITION :

• DNA Microarray is a technique for genome Analysis , where thousands of genes are analysed at a time in a single experiment simultaneously to determine which genes are expressed in a particular sample

• These experiments rely on the principle of hybridisation

• Thousands of known probes are fabricated onto a small solid slide (Glass,Silicon ,Nylon) in a particular order as spots and called DNA microarray or DNA chip.

• The spots can be DNA , cDNA , or oligonucleotide.

CONTD. HISTORY:• Traditionally, measurement of gene activity was

performed as one gene analysed in many samples on blots using the northern hybridisation and differential display.

• cDNA microarray was first introduced by patrick browns lab in 1995.

• Oligomicroarray was introduced by Affimatrix in 1996.

PRINCIPLE :• The principle of DNA microarray

technology is based on the fact that complimentary sequences of DNA can be used to hybridise , immobilised DNA molecules.

• There are four major steps in performing a typical microarray experiment.

MICROARRAY EXPERIMENT

TYPES OF MICROARRAY There are major two types of Microarray :

cDNA MicroarrayOligonucleotide Microarray

cDNA Microarray:

cDNA Microarray ,first introduced by Patrick Brown ,is also termed as spotted microarray

In this technique ,DNA fragments (genes) are amplified by PCR and then spotted onto a glass slide in a grid like pattern.

This is a two-channeled microarray that is typically hybridised with cDNA from two samples to be compared on the same slide for visualisation of genes.

These samples are flourescently labelled (Cys 3-rhodamine for red and Cys 5-fluorescin for green) for easy detection.

Data from a cDNA microarray provides information about the relative expression of genes in two different biological samples.

Oligonucleotide Microarray: In this method, a DNA microarray slide is fabricated by

synthesising 11-16 short oligonucleotides (approx. 25bp in length) at very high density for each expressed gene on a slide.

Here oligonucleotides are synthesised at fixed locations by photolithographic technique and chemical synthesis technique.

In Affimatrix, a 25 bp long oligos are taken and a total of 100 light masks are made for each base at each layer.

An oligo-MA contains two sets of probes for each gene One set for perfect match and another that contains mismatch nucleotides.

Here two or more samples can be compared at a time but each sample needs different microarray slides.

These arrays are used in genotyping experiments, where sequences of alternate gene forms are synthesised to detect polymorphisms and mutations.

A white spot is generated for strong hybridisation and no hybridisation produces black spots.

In between these two colours is marginal hybridisation. Oligonucleotide microarrays avoid the cross-

hybridisation that may occur with the cDNA microarray. They are much expensive and also need very accurate

gene annotation to fabricate match and mismatch probes.

Contd.

AFFIMETRIX GENE CHIP EXPERIMENT

AFFIMETRIX ARRAYS

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

below: Gene expression profiling Discovery of drugs Diagnostics and genetic engineering in proteomics Functional genomics DNA sequencing Toxicological research.

Provide 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.

ADVANTAGES:

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.

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

DISADVANTAGE: