Introduction to Gene Expression,

& Microarray Technology

Gene expression

• Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product.

• These products are often proteins

Also known as DNA Chip

• Allows simultaneous measurement of the level of transcription for every gene in a genome (gene expression)

• Are small, solid supports onto which the sequences or subsequences from thousands of different genes are attached.

• The supports are usually glass microscope slides, or silicon chips or nylon membranes. The DNA is printed, spotted, or actually synthesized directly onto the support.

• The spots can be DNA, cDNA, or oligonucleotides

• It consists of an arrayed series of thousands of microscopic spots of DNA oligonucleotides, each containing specific DNA sequence.

DNA microarrays are created by spotting every gene in a genome onto a glass microscope slide.

Modified from http://darwin.bio.uci.edu/~faculty/wagner/array2.html

Each spot represents different gene/clone

Why Are Microarrays Important?

• Microarrays are a significant advance both because they may contain a very large number of genes and because of their small size.

• Microarrays are therefore useful when one wants to survey a large number of genes quickly or when the sample to be studied is small.

• .

The process

Sample preparation

• The two samples to be compared .

• In this example treated sample (case) and untreated sample (control).

DNA probe

• A short sequence of DNA labelled that is used for the detection of a complementary nucleotide sequence.

Hybridization

cover

slip

Hybridize for

5-12 hours

Binding of cDNA target samples to cDNA probes on the slide

DNA microarrays: step by step

• Production of DNA probes

• Printing or “spotting” Printing or “spotting”

Ngai Lab arrayer , UC Berkeley

The arrayer

Print-tip head

• Once extracted, the mRNAs need to be labelled with fluorescent markers1 so that they can be detected later, on the surface of the micorarray.

• mRNA of the control cells is usually labelled with green fluorescent marker, and mRNA of the cells under study with red fluorescent marker.

• the control mRNA (labelled green) is mixed with the test mRNA (labelled red).

• The mixture is then flooded over the surface of a slide, which is then incubated at 42°C,

• After 12 hours, the microarray is washed .

• The microarray is now ready for scanning.

Labeled DNA hybridizes to corresponding DNA/gene

ScanningDetector

PMT

Image

Duplicate spots

• But wait a minute there are not just red and green dots there are yellow dots as well!

• This can easily be explained.

• It is clear that the red spots contain mRNA from cancer cells and green spots mRNA from noncancerous control cells.

• yellow! Remember mRNA hybridizes with its complementary DNA and one spot on the microarray represents billions of copies of DNA from ONE gene.

• In other words, when a spot is yellow, there are equals amounts of mRNA of the gene found in cancerous and control cells.

• And black means that there is no mRNA of that gene either in the control or cancerous cells.

RGB overlay of Cy3 and Cy5 images