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Chapter 20

Chapter 20. DNA Technology and Genomics. Overview: Understanding and Manipulating Genomes. Sequencing of the human genome was largely completed by 2003 DNA sequencing has depended on advances in technology, starting with making recombinant DNA

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Chapter 20

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  1. Chapter 20 DNA Technology and Genomics

  2. Overview: Understanding and Manipulating Genomes • Sequencing of the human genome was largely completed by 2003 • DNA sequencing has depended on advances in technology, starting with making recombinant DNA • In recombinant DNA, nucleotide sequences from two different sources, often two species, are combined in vitro into the same DNA molecule • Methods for making recombinant DNA are central to genetic engineering, the direct manipulation of genes for practical purposes

  3. DNA technology has revolutionized biotechnology, the manipulation of organisms or their genetic components to make useful products • An example of DNA technology is the microarray, a measurement of gene expression of thousands of different genes

  4. Concept 20.1: DNA cloning permits production of multiple copies of a specific gene or other DNA segment • To work directly with specific genes, scientists prepare gene-sized pieces of DNA in identical copies, a process called gene cloning

  5. DNA Cloning and Its Applications: A Preview • Most methods for cloning pieces of DNA in the laboratory share general features, such as the use of bacteria and their plasmids • For cloning genes, a plasmid is first isolated from a bacterial cell, then the foreign DNA is inserted into it • Results in recombinant DNA molecule • Cloned genes are useful for making copies of a particular gene and producing a gene product

  6. LE 20-2 Cell containing gene of interest Bacterium Gene inserted into plasmid Bacterial chromosome Plasmid Gene of interest Recombinant DNA (plasmid) DNA of chromosome Plasmid put into bacterial cell Recombinant bacterium Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Protein expressed by gene of interest Gene of interest Copies of gene Protein harvested Basic research and various applications Basic research on gene Basic research on protein Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Human growth hor- mone treats stunted growth

  7. Using Restriction Enzymes to Make Recombinant DNA • Bacterial restriction enzymes cut DNA molecules at DNA sequences called restriction sites • A restriction enzyme usually makes many cuts, yielding restriction fragments • The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “sticky ends” that bond with complementary “sticky ends” of other fragments • DNA ligase is an enzyme that seals the bonds between restriction fragments

  8. LE 20-3 Restriction site 5¢ 3¢ DNA 3¢ 5¢ Restriction enzyme cuts the sugar-phosphate backbones at each arrow. Sticky end DNA fragment from another source is added. Base pairing of sticky ends produces various combinations. Fragment from different DNA molecule cut by the same restriction enzyme One possible combination DNA ligase seals the strands. Recombinant DNA molecule

  9. Animation: Restriction Enzymes

  10. Cloning a Eukaryotic Gene in a Bacterial Plasmid • In gene cloning, the original plasmid is called a cloning vector • A cloning vector is a DNA molecule that can carry foreign DNA into a cell and replicate there

  11. Producing Clones of Cells • Cloning a human gene in a bacterial plasmid can be divided into six steps: 1. Vector and gene-source DNA are isolated 2. DNA is inserted into the vector 3. Human DNA fragments are mixed with cut plasmids, and base-pairing takes place 4. Recombinant plasmids are mixed with bacteria 5. The bacteria are plated and incubated 6. Cell clones with the right gene are identified Animation: Cloning a Gene

  12. LE 20-4_1 Bacterial cell lacZ gene (lactose breakdown) Human cell Isolate plasmid DNA and human DNA. Restriction site ampR gene (ampicillin resistance) Bacterial plasmid Gene of interest Sticky ends Human DNA fragments Cut both DNA samples with the same restriction enzyme. Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids. Recombinant DNA plasmids

  13. LE 20-4_2 Bacterial cell lacZ gene (lactose breakdown) Human cell Isolate plasmid DNA and human DNA. Restriction site ampR gene (ampicillin resistance) Bacterial plasmid Gene of interest Sticky ends Human DNA fragments Cut both DNA samples with the same restriction enzyme. Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids. Recombinant DNA plasmids Introduce the DNA into bacterial cells that have a mutation in their own lacZ gene. Recombinant bacteria

  14. LE 20-4_3 lacZ gene (lactose breakdown) Bacterial cell Human cell Isolate plasmid DNA and human DNA. Restriction site ampR gene (ampicillin resistance) Bacterial plasmid Gene of interest Sticky ends Human DNA fragments Cut both DNA samples with the same restriction enzyme. Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids. Recombinant DNA plasmids Introduce the DNA into bacterial cells that have a mutation in their own lacZ gene. Recombinant bacteria Plate the bacteria on agar containing ampicillin and X-gal. Incubate until colonies grow. Colony carrying recombinant plasmid with disrupted lacZ gene Colony carrying non- recombinant plasmid with intact lacZ gene Bacterial clone

  15. Identifying Clones Carrying a Gene of Interest • A clone carrying the gene of interest can be identified with a nucleic acid probe having a sequence complementary to the gene • This process is called nucleic acid hybridization • An essential step in this process is denaturation of the cells’ DNA, separation of its two strands

  16. LE 20-5 Colonies containing gene of interest Master plate Master plate Probe DNA Radioactive single-stranded DNA Solution containing probe Gene of interest Film Single-stranded DNA from cell Filter Filter lifted and flipped over Hybridization on filter A special filter paper is pressed against the master plate, transferring cells to the bottom side of the filter. The filter is treated to break open the cells and denature their DNA; the resulting single-stranded DNA molecules are treated so that they stick to the filter. The filter is laid under photographic film, allowing any radioactive areas to expose the film (autoradiography). After the developed film is flipped over, the reference marks on the film and master plate are aligned to locate colonies carrying the gene of interest.

  17. Storing Cloned Genes in DNA Libraries • A genomic library that is made using bacteria is the collection of recombinant vector clones produced by cloning DNA fragments from an entire genome • A genomic library that is made using bacteriophages is stored as a collection of phage clones

  18. LE 20-6 Foreign genome cut up with restriction enzyme or Bacterial clones Recombinant plasmids Phage clones Recombinant phage DNA Plasmid library Phage library

  19. A complementary DNA (cDNA) library is made by cloning DNA made in vitro by reverse transcription of all the mRNA produced by a particular cell • A cDNA library represents only part of the genome—only the subset of genes transcribed into mRNA in the original cells

  20. Cloning and Expressing Eukaryotic Genes • As an alternative to screening a DNA library, clones can sometimes be screened for a desired gene based on detection of its encoded protein • After a gene has been cloned, its protein product can be produced in larger amounts for research

  21. Bacterial Expression Systems • Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells • To overcome differences in promoters and other DNA control sequences, scientists usually employ an expression vector, a cloning vector that contains a highly active prokaryotic promoter

  22. Eukaryotic Cloning and Expression Systems • The use of cultured eukaryotic cells as host cells and yeast artificial chromosomes (YACs) as vectors helps avoid gene expression problems • YACs behave normally in mitosis and can carry more DNA than a plasmid • Eukaryotic hosts can provide the posttranslational modifications that many proteins require

  23. One method of introducing recombinant DNA into eukaryotic cells is electroporation, applying a brief electrical pulse to create temporary holes in plasma membranes • Alternatively, scientists can inject DNA into cells using microscopic needles • Once inside the cell, the DNA is incorporated into the cell’s DNA by natural genetic recombination

  24. Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR) • The polymerase chain reaction, PCR, can produce many copies of a specific target segment of DNA • A three-step cycle—heating, cooling, and replication—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules

  25. LE 20-7 5¢ 3¢ Target sequence Genomic DNA 3¢ 5¢ 5¢ 3¢ Denaturation: Heat briefly to separate DNA strands 3¢ 5¢ Annealing: Cool to allow primers to form hydrogen bonds with ends of target sequence Cycle 1 yields 2 molecules Primers Extension: DNA polymerase adds nucleotides to the 3¢ end of each primer New nucleo- tides Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence

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