BIOM 121 1st Edition Lecture 4 Outline of Current Lecture 1. Microarray2. Next generation sequencing3. Function of protein4. Inactivating Current LectureMicroarray- study the of expression of genes and simultaneously look at mRNA levels1. Have 1 set of cultured cells plan, and another have them with serum (drug)2. Isolate mRNA form both sets of cells and convert it to cDNA and label with fluorescent dye3. Mix drug cDNA and no drug cDNA. Where the two colors cancel each other out, they arethe exact same. If have excess of drug dye, then more drug mRNA is expressed.Next Generation Sequencing – looking at global changes, sequence every single mRNA 1. Take mRNA from sample and attach primer on each end2. Attach to cDNATells you how much mRNA is there, and how much.Function of a ProteinOverexpress the gene in Animal Cells: Goal is to make a lot of proteins- when cloning in bacteria, only needed a plasmid because both replicate. But animal cells are different. A. Transient Transfection= cDNA (gene x) and viral origin of replication.a. Doesn’t last long, cells try to destroy virusb. No passage into daughter cells, but make lots of proteins fastB. Stable Transfection= gets gene to integrate into chromosome by crossing overThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.a. Insert cDNA (gene x) into vectorb. Put cell into petri dish and 1 out of 10,000 times it will cross over into other chromosomesc. Becomes part of DNA and replicates lots of protein madeC. Western Blot- measure level of proteina. Once know that it makes lots of resistance protein, then measure the functionInactivating- function of a gene in Eukaryotes; no protein is made; see what happens if protein is not presentChapter 7Transcriptional Control of Gene ExpressionA. Common transcription elements in all Eukaryotic genes:a. TATA box: upstream; rich in TA’s on genes that are actively transcribedb. Initiator= start sitec. CpG island- 60-70% of genes; area rich in CG’s; RNA polymerase looking for this to have start
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