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Lecture31 (Brandl8).docx

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Western University
Biochemistry 2280A
Eric Ball

BrandlLecture 8 NotesProblem You have found a gene YFG that you hypothesize is important for the development of muscleQuestions 1 Is this gene expressed in muscle 2 In what other tissues cell types is the gene expressed Steps in a Northern BlotStep 1isolate RNA from muscle tissue and a group of other tissues to be examined eg muscle liver spleen brain o Note to partially purify mRNA the RNA is often first bound to a oligodT column then elutedStep 2separate the RNA on the basis of size by agarose gel electrophoresis o Still see 2 major bandsribosomal RNA so even if you try to purify the mRNA you always see these 2 bands Step 3transfer the separated RNA to nitrocellulose nylon membrane BLOTTING o Result of blotting is that you get an exact replica of the RNA that was on the gelStep 4radioactively labeled DNA probe that encodes YFG o If the probe is double stranded DNA it must be denatured o If it singlestranded DNA or RNA it must be the complementary strand o Radioactive probe complementary to that RNA o Use a hot probe Step 5incubate the HOT probe with the filter to allow complementary strands to anneal hybridizeo 10 C below the Tmo Remove nitrocellulose paper with tightly bound nucleic acids radioactive labeled probe hybridized to separate DNA o Have a sealed plastic bag with labeled probe in buffer Step 6wash away the nonspecifically bound probeStep 7Expose the probed filter to Xray film to determine where the hot probe has annealedo There will be various films on the xray of the blotted membraneo Radioactive probe will appear as bands in the lanes in which YFG is expressed YFG is expressed in muscle And also in the liverWhy might there be 2 bands in the Liver sampleMicroarraysDNA microarrays allow the analysis of thousands of genes at a time In this procedure DNA probes labeled with fluorescent tags are hybridized to DNA fragments fixed to microscopic slides o Allows the comparative analysis of thousands of genes at the same time o 25000 spots of DNA on that glass slideThe standard use of microarrays is to answer the question of which genes are induced in a cell type in response to a change in condition
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