DNA Microarray - Montana State University：基因芯片-蒙大拿州立大学.ppt

Jamie MashekWhat we will be discussing…?What is DNA microarray??The purpose of using DNA microarray.?The plate.?Steps to perform a microarray.?Benefits.? is DNA Microarray??Scientists used to be able to perform ic analyses of a few genes at once. DNA microarray allows us to analyze thousands of genes in one experiment!Purposes.?So why do we use DNA microarray??To measure changes in gene expression levels – two samples’ gene expression can pared from different samples, such as from cells of different stages of mitosis.?To observe genomic gains and losses. parative Genomic Hybridization (CGH)?To observe mutations in Plate.?Usually mercially.?Made of glass, silicon, or nylon.?Each plate contains thousands of spots, and each spot contains a probe for a different gene.?A probe can be a cDNA fragment or a synthetic oligonucleotide, such as BAC (bacterial artificial chromosome set).?Probes can either be attached by robotic means, where a needle applies the cDNA to the plate, or by a method similar to making silicon chips puters. The latter is called a Gene ’s perform a microarray!1)Collect )Isolate )Create Labelled ))Microarray )Analyze 1: Collect Samples.?This can be from a variety anisms. We’ll use two samples – cancerous human skin tissue & healthy human skin tissueSTEP 2: Isolate mRNA.?Extract the RNA from the samples. Using either a column, or a solvent such as phenol-chloroform.?After isolating the RNA, we need to isolate the mRNA from the rRNA and tRNA. mRNA has a poly-A tail, so we can use a column containing beads with poly-T tails to bind the mRNA.?Rinse with buffer to release the mRNA from the beads. The buffer disrupts the pH, disrupting the hybrid 3: Create Labelled DNA.?Add a labelling mix to the RNA. The labelling mix contains poly-T (oligo dT) primers, reverse transcriptase (to make cDNA), and fluorescently dyed nucleotides.?We will add cyanine 3 (fluoresces