Transcript Arrays
The Human Genome Project and ~ 100 other genome projects: • Done!! • What do you find?? • predictions of all genes/protein sequences. What’s next?? Brief History of Molecular Genetics • Pre-Genomic Era- Study of Individual Genes using techniques of Molecular Biology. • Genomic Era- Sequencing of entire genomes. Analytical techniques include Sequencing and Gene Finding. The new way of doing biologyPost-Genomic Era • Genome Analysis of Gene Expression. Monitoring activity (turn on, turn off) of all genes together. • #1 tool- DNA microarrays Microarray Technology? What is it?? The future . . . • DNA microarrays (also called DNA chips or Gene chips) are miniaturized laboratories for the study of gene expression. • Microarrays are small pieces of glass (or silicon or nylon) about 2 or 3 cm square, coated with DNA. • The DNA is spotted by high speed robots in a very precise pattern. The droplets of DNA (measured in pL or nL) are separated by _x_ microns. • The surface of the array is covered with thousands, tens of thousands, (or soon with hundreds of thousands) of spots, each spot containing a different DNA oligomer. • DNA Microarray Technique (animatedrequires Flash plug-in) • Each oligomer in a DNA microarray can serve as a probe to detect a unique, complementary DNA or RNA molecule. Many microarray experiments have been performed with Yeast. • Genome is completely sequenced and well annotated. • Select a PCR primer pair that amplifies each ORF. • My arrays- all yeast genes represented (70mers). PCR primers for sale . . . • To our sales list we add Yeast ORF specific primers for over 6,000 ORFs! These primers have been designed to amplify, from genomic DNA, the complete coding region including the start and stop codons. For yeast this is possible as very few yeast genes contain introns. Common Protocol In yeast (or other single-celled organisms) compare cells grown in two different conditions. • Why? What information are biologists searching for? • GCAT Animation See Microarray Data • The Stanford Microarray Database microarray data storage, retrieval, analysis, visualization, and database software There are two distinct way of making DNA arrays: • DNA microarrays: Also referred to as "microarrays." Non- porous solid supports, such as glass have facilitated miniaturization and fluorescence based detection. About 10,000 cDNAs can be robotically spotted onto a microscope slide and hybridized with a double labeled probe, using protocols pioneered by Pat Brown and colleagues at Stanford. Gene Chips or DNA Chips • Oligomers synthesized directly on the chips (pioneered by Affymetrix). GeneChip ® is an Affymetrix product where they have adapted photolithographic masking techniques used in semiconductor manufacture to produce arrays with 400,000 distinct oligonucleotides. Affymetrix – Gene Chips • DNA is short (25 nt) • Small variances (SNPs) can be detected • Only one Dye can be used at a time • • • • DNA Microarrays cDNA is spotted on glass plates cDNA is from 100nt to 2kb in length Very efficient hybridization Small variances (SNPs) can’t be seen #1 use of Microarrays- Investigate Changes in Gene Expression. Compare cells before and after perturbation: • • • • • Nutritional Environmental Disease Toxin Different Stages of Development Human Cells • Compare normal tissue vs. diseased tissue. • DNA Microarray Technique (animatedrequires Flash plug-in) • PNAS -- article Researchers love DNA chips; Doctors will soon love DNA chips • Researchers love DNA chips because they give a huge amount of information, fast, at low cost. • Doctors will soon learn to love them because there are many times when a doctor would like to know something about a patient's genes (such as whether the patient is likely to respond well to a certain drug). When the price comes down enough, microarrays will likely become routine tools in the doctor's office. Clinical Applications? • If this research pays off, scientists and doctors will see a new, more informative face of cancer for individual tumors. That could produce tremendous gains in cancer care. • Doctors could better match patients to the best treatment. That could improve the outcome and spare patients the side effects of therapies that won't help. • Science -- Putting Gene Arrays to the Test • Science -- Revolution in Diagnostics • Since the development of DNA microarray technology in the late 1990s, it has become apparent that the increase in available gene expression data will eventually parallel the growth of the sequence and structure databases.