LS15GeneexpressionAnanyalsis

LS15GeneexpressionAnanyalsis - Gene Expression Analysis...

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Gene Expression Analysis
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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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S o uthe rn Blo t to  De te c t S pe c ific  DNAs  (No rthe rn  Blo ts  fo r RNAs )
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  Southern blots can detect specific DNA fragment(s) in complex mixtures of restriction  fragments separated by gel electrophoresis
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Gene copy number estimated by Southern blot Neu oncogene is amplified in ~30% of  with poor clinical prognosis Experiment: Southern blot comparing  genomic DNA samples probed for Neu Breast cancer sample with amplified Neu  shows more intense hybridized band vs  Recombinant antibody against Neu  protein (Herceptin) from Genentech now  used to successfully treat breast cancers  with high Neu Elevated Neu expression is important  diagnostic tool for predicting patient  responses to Herceptin (early example of  personalized medicine)   Normal Tissue Tumor Tissues Amplified Neu
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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 deletion/insertion may have different restriction map than the wild type (normal) gene - Point mutation (single nucleotide change) DNA with single nucleotide differences can also be distinguished by Southern blot analysis
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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
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Southern blotting can reveal mutations in a gene. cDNA, no Eco RI sites
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LS15GeneexpressionAnanyalsis - Gene Expression Analysis...

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