Introduction to Gene Expression, & Microarray Technology

1. Introduction to Gene Expression,& Microarray Technology

2. 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

4. Also known as DNA Chip • Allows simultaneous measurement of the level of transcription for every gene in a genome (gene expression)

5. 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

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

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

10. Each spot represents different gene/clone Modified fromhttp://darwin.bio.uci.edu/~faculty/wagner/array2.html

12. 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. • .

13. The process

20. Sample preparation

21. The two samples to be compared . • In this example treated sample (case) and untreated sample (control).

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

25. Hybridization cover slip Hybridize for 5-12 hours Binding of cDNA target samples to cDNA probes on the slide

26. DNA microarrays: step by step • Production of DNA probes • Printing or “spotting” Printing or “spotting”

27. The arrayer Ngai Lab arrayer , UC Berkeley Print-tip head

31. 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.

33. 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.

37. Labeled DNA hybridizes to corresponding DNA/gene

38. Image Duplicate spots Scanning Detector PMT

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

42. 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.

44. 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.

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

48. RGB overlay of Cy3 and Cy5 images