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Chapter 20

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.

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Chapter 20

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  1. Chapter 20 DNA Technology and Genomics

  2. DNA Cloning

  3. Restriction Enzymes Restriction Enzymes are found in bacterial cells. When they cut DNA at a specific place, they leave sticky ends.

  4. Cloning • The cloning vector is the original plasmid. • After insertion of the gene of interest into the plasmid the plasmid is called recombinant DNA.

  5. Identifying the gene of interest Use different probes How would you identify different genes?

  6. 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

  7. cDNA Library

  8. Different libraries have different uses • Which library would you use in the following scenarios? • You want to clone a gene, without knowing where and how it is expressed. • You want to know regulatory sequences. • You want to know the coding sequences of a gene. genomic library genomic library cDNA library

  9. 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. Expressing Eukaryotic Genes

  10. 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

  11. Thermocycler for PCR

  12. Polymerase Chain Reaction (PCR)

  13. Gel Electrophoresis

  14. RFLP - made from noncodingDNA 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.

  15. Homologous segments of DNA from a family in which some members have a genetic disease

  16. Figure 20.x1a Laboratory worker reviewing DNA band pattern

  17. Figure 20.x1b DNA study in CDC laboratory

  18. Southern Blotting Techniqueused to detect specific DNA sequences

  19. Restriction fragment analysis by Southern blotting

  20. 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

  21. Francis Collins, Former Director of the Human Genome Project

  22. Mapping the Genome Ordering of genetic markers such as RFLPs ordering of large overlapping fragments cloned in YAC or BAC vectors

  23. Shotgun Approachused by J. Craig Venter and Celera Genomics

  24. Sanger Sequencing

  25. 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”

  26. Genome Sizes

  27. Genomes • If there is no pattern between complexity and number of genes what makes organisms more complex? • More involved regulation • Alternative splicing of RNA

  28. 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

  29. Genome Wide Expression Resulting colors shows which genes were being expressed in each tissue Uses two tissue samples with differently colored fluorescent nucleotides

  30. 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

  31. 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)

  32. DNA microarray assay for gene expression

  33. Practical Applications • Medical • Disease diagnosis • Gene Therapy • Medicines • Forensics • Environmental Cleanup • Agriculture • Pharm animals • GMO foods/plants

  34. Pharm Animals

  35. Gene Therapy

  36. Making Transgenic Plants

  37. “Golden” rice contrasted with ordinary rice

  38. The process of separating • DNA fragments to analyze them • is called. .. 2) Why does the DNA migrate in the gel box?

  39. 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:

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