LS15GeneexpressionAnanyalsis

LS15GeneexpressionAnanyalsis - Lecture 15: Gene Expression...

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1 Lecture 15: Gene Expression Analysis Many methods for gene expression analysis Southern blots Determine gene structure & genomic organization (e.g., DNA rearrangements, deletions, insertions) map transcription control regions, detect mutations, analyze gene copy number, DNA fingerprinting, etc. Northern blots Analyze mRNA species & RNA processing events, determine changes in mRNA splicing in biological processes. Polymerase chain reaction (PCR) analysis Analyze quantitative and semi- quantitative amounts of DNA and RNA, detect mutations, generate mutations, amplify DNA for cloning. Microarray analysis Analyze quantitative changes in different mRNAs expressed from up to every gene in a genome - can be used to define global gene expression patterns in biological processes Proteomic analysis Analyze quantitative changes in different protein expressed from up to every gene in a genome - can be used to define global gene expression patterns in biological processes
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2 Southern Blot to Detect Specific DNAs (Northern Blots for RNAs) Southern blots can detect specific DNA fragment(s) in complex mixtures of restriction fragments separated by gel electrophoresis
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3 Gene copy number estimated by Southern blot Neu oncogene is amplified in ~30% of advanced breast cancers & is correlated with poor clinical prognosis Experiment: Southern blot comparing genomic DNA samples probed for Breast cancer sample with amplified shows more intense hybridized band vs Recombinant antibody against Neu protein (Herceptin) from Genentech now used to successfully treat breast cancers with high Neu •E l v a t e d Neu expression is important diagnostic tool for predicting patient responses to Herceptin (early example of personalized medicine) Normal Tissue Tumor Tissues Amplified Neu An example of the application of Southern -Detection of mutations by Southern blot analysis -More than 3000 different human genetic diseases are caused by mutations in single genes. These mutations may be caused by DNA deletions/insertion, point mutations, etc. - Deletion/insertion mutation This type of mutation can be detected using Southern blot analysis because a mutant gene containing a
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4 Southern Blotting can be used to compare genomic structure to a cDNA cDNA, no Eco RI sites label Genomic DNA cut with Eco RI Southern blot probed with labeled cDNA 1 2 3 Genomic DNA RI RI RI RI 1 2 3 1 2 3 1 2 3 1 2 3 Southern blotting can reveal mutations in a gene. cDNA, no Eco RI sites
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LS15GeneexpressionAnanyalsis - Lecture 15: Gene Expression...

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