introduction to gene expression, & microarray technology
DESCRIPTION
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. - PowerPoint PPT PresentationTRANSCRIPT
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