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Chapter 20 DNA Technology and Genomics DNA Cloning Restriction Enzymes Restriction Enzymes are found in bacterial cells. When they cut DNA at a specific place, they leave sticky ends. Cloning The cloning vector is the original plasmid. After insertion of the gene of interest into the plasmid the plasmid is called recombinant DNA. Identifying the gene of interest Usewould different How youprobes identify different genes? DNA Libraries Shotgun approach makes thousands of different recombinant plasmids This set of plasmid clones is called a Besides using plasmids scientists use as cloning vectors cDNA Library Different libraries have different uses 1. Which library would you use in the following scenarios? You want to clone a gene, without knowing where and how it is expressed. genomic library 2. You want to know regulatory sequences. genomic library 3. You want to know the coding sequences of a gene. cDNA library Expressing Eukaryotic Genes How can scientists get bacterial cells to express eukaryotic genes? Insert the desired gene with a prokaryotic promoter attached – this is called an expression vector How do they remove introns? Insert cDNA in the first place. YACs can also be used since they are originally from eukaryotic cells. DNA Amplification PCR = polymerase chain reaction Can make billions of copies of a target segment of DNA in a few hours can use scanty or impure sources of DNA (Ex. the blood at the O.J. Simpson crime scene) can be used to identify bacteria, viruses, and genetic sequences for diseases Thermocycler for PCR Polymerase Chain Reaction (PCR) Gel Electrophoresis RFLP - made from noncoding DNA resulting from differences in the restriction sites on homologous chromosomes Used to determine where genes are located and where they are expressed RFLPs are found on noncoding segments of DNA and are due to VNTRs. Homologous segments of DNA from a family in which some members have a genetic disease Figure 20.x1a Laboratory worker reviewing DNA band pattern Figure 20.x1b DNA study in CDC laboratory Southern Blotting Technique used to detect specific DNA sequences Restriction fragment analysis by Southern blotting Entire genomes can be mapped at the DNA level •Human Genome Project started in 1990 Rough Draft – 2000; completed 2003 •James Watson – original director •Francis Collins – director when it was finished; now at NIH •J. Craig Venter – Celera Genomics Francis Collins, Former Director of the Human Genome Project Ordering of genetic markers such as RFLPs Mapping the Genome ordering of large overlapping fragments cloned in YAC or BAC vectors Shotgun Approach used by J. Craig Venter and Celera Genomics Sanger Sequencing Identifying Protein Coding Genes What would you look for if you wanted to find an unknown protein coding gene? Scientists use computers to search for short coding sequences similar to those present in known genes. these are called “express service tags” Genome Sizes Genomes If there is no pattern between complexity and number of genes what makes organisms more complex? More involved regulation Alternative splicing of RNA Determining Gene Function How can we determine the function of different genes? RNAi; silence a gene and see what it did Insert synthetic double stranded RNA’s that match a gene that will inactivate translation This was used to identify the function of C. elegans genes Genome Wide Expression Resulting colors shows which genes were being expressed in each tissue Uses two tissue samples with differently colored fluorescent nucleotides What is a DNA Microarray? •allows scientists to perform an experiment on thousands of genes at the same time •Each spot on a microarray contains multiple identical strands of DNA •each DNA sequence is unique •each spot represents one gene Comparing Genomes Using Microarrays Has revealed many similar regions even between yeast and us. Can be used to derive evolutionary relatedness. Can use differences between closely related species to explain phenotypic differences Has led to proteomics (study of full protein sets) DNA microarray assay for gene expression Practical Applications Medical Disease diagnosis Gene Therapy Medicines Forensics Environmental Cleanup Agriculture Pharm animals GMO foods/plants Pharm Animals Gene Therapy Making Transgenic Plants “Golden” rice contrasted with ordinary rice 1)The process of separating DNA fragments to analyze them is called. .. 2) Why does the DNA migrate in the gel box? 3) Name two other uses of DNA fingerprinting that are not related to crime scenes. 4) The different DNA fragments that are formed by a fingerprint are called: