Reverse Transcription PCR (RT-PCR) - mcdb逆转录PCR（RT-PCR）- MCDB.pptVIP

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2018-04-17 发布于江西
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Reverse Transcription PCR (RT-PCR) - mcdb逆转录PCR（RT-PCR）- MCDB.ppt

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Reverse Transcription PCR (RT-PCR) - mcdb逆转录PCR（RT-PCR）- MCDB.ppt

Gene Regulation: What it is, and how to detect it By Jordan, Jennifer, and Brian What is the Promoter Region of a Gene? How Can the Gene Promoter Region be Located and Isolated? Location: The vital region of the promoter is somewhere upstream from the gene of interest, and downstream from the “stop” codon directly before the gene of interest. Knowing that the region of interest is somewhere between these two points is all that is necessary! Isolation If the entire genome of the organisms has been sequenced (as is the case with arabidopsis), then the exact nucleotide sequences of these areas is known. From the sequence information primers can be designed and the gene promoter fragment can be amplified by the polymerase chain reaction (PCR). How can an isolated promoter region show where a gene is active? A previously amplified promoter region (as described in the slide prior) can be fused to a GFP reporter gene. The fused region can then be transformed back into the original organism’s genomic DNA. Observing where the green fluorescence is present will visualize where the promoter region is active in the original organisms. Isolate RNA from Plant Synthesize cDNA Using RT Amplify cDNA Using Primers Visualize with Gel Electrophoresis Presence of mRNA Starting point for determining expression Expression of gene determined by observing proteins Can only determine transcription GeneChip Analysis Using cDNA created from the mRNA isolated from various organs, we can analyze the mRNA accumulation levels for all genes. This is done by creating the complementary strands of the known gene sequences and assembling them on a chip. The sequences are tagged with flourescent tags that glow a certain color when in contact with the complementary strand. This is what you get as a resulting image: We can then translate the data into graphs like * Upstream region of a gene, which regulates when/where the gene is to be transcribed. Essentially the promoter region acts as the “on