Lecture15 (3)

Lecture15 (3) - Lecture15: RecombinantDNA GenomicAnalysis...

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Lecture 15: Recombinant DNA Genomic Analysis
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Parental Imprinting Genes are expressed differently depending upon the parent of origin. For imprinted genes, expression occurs from only one parents’ allele - the other parents’ allele is silenced. The silenced DNA is marked in a parent-of-origin- specific manner by DNA methylation or other epigenetic mechanisms
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methylated methylated CG island CG island closed chromatin Imprinted genes may be cytosine methylated, and have altered chromatin structure CG island CG island “relaxed” chromatin non-imprinted gene imprinted gene transcription no transcription
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In the germ-line , imprints are erased and reestablished according to the sex of the individual FIGURE 11.21
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Imprinting and Human Disorders Angelman syndrome Angelman syndrome Prader-Willi syndrome Prader-Willi syndrome Both can be caused by the same deletion on chr 15 although they have different phenotypes.
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LECTURE 16: Recombinant DNA, Genomics OUTLINE: Recombinant DNA technology : Applications of PCR Genomics Genome sequencing Gene expression profiling Systems biology and functional genomics Personal Genomics
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A lesson in practical molecular biology: express human topoisomerase I in  S. cerevisiae   to test new drugs cheaply - Design a cloning strategy what are the elements within a plasmid? choose restriction enzymes - Amplify your gene using PCR - Clone your gene into a plasmid - Sequence your gene - Analyze transformed yeast with a Southern blot - Verify mRNA and protein expression
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camptothecin topoisomerase I Aim: identify new inhibitors of human  topoisomerase I by screening in yeast
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DNA gyrase (topisomerase) removes extra twists
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camptothecin topoisomerase I Clone the human topoisomerase I gene and  express it in  S. cerevisiae
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Plasmid Cloning Vectors 1) replication origin 2) selectable marker 3) polylinker/ multiple cloning site DNA ligase Clone = DNA in a vector that can be replicated to make many identical copies
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Many restriction enzymes produce ends with short, single-stranded overhangs. Complementary “sticky-ends” allow efficient cloning of DNA fragments into plasmids Polylinker/multiple cloning site = region of a vector with many restriction enzyme cleavage sites Cloning an insert into a plasmid
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Restriction Enzymes Enzyme    Source      Recognition Sequence     Cut                      Eco RI Escherichia 
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Lecture15 (3) - Lecture15: RecombinantDNA GenomicAnalysis...

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