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Chapter 20

Chapter 20. Biotechnology. Why biotechnology?. Diagnosis of disease Gene therapy Pharmaceutical development Forensic applications Environmental remediation Agricultural applications. Overview: The DNA Toolbox. Sequencing of the human genome was completed by 2007

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Chapter 20

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  1. Chapter 20 Biotechnology

  2. Why biotechnology? • Diagnosis of disease • Gene therapy • Pharmaceutical development • Forensic applications • Environmental remediation • Agricultural applications

  3. Overview: The DNA Toolbox • Sequencing of the human genome was completed by 2007 • 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

  4. Methods for making recombinant DNA are central to genetic engineering, the direct manipulation of genes for practical purposes • 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

  5. Fig. 20-1

  6. Concept 20.1: DNA cloning yields multiple copies of a gene or other DNA segment • To work directly with specific genes, scientists copy pieces of DNA, a process called DNA cloning

  7. Gene cloning involves using bacteria to make multiple copies of a gene • Foreign DNA is inserted into a plasmid, and the recombinant plasmid is inserted into a bacterial cell • Reproduction in the bacterial cell results in cloning of the plasmid including the foreign DNA • This results in the production of multiple copies of a single gene

  8. Fig. 20-2 Cell containing geneof interest Bacterium 1 Gene inserted intoplasmid Bacterialchromosome Plasmid Gene ofinterest RecombinantDNA (plasmid) DNA of chromosome 2 Plasmid put intobacterial cell Recombinantbacterium 3 Host cell grown in cultureto form a clone of cellscontaining the “cloned”gene of interest Gene ofInterest Protein expressedby gene of interest Copies of gene Protein harvested Basic research andvarious applications 4 Basicresearchon protein Basicresearchon gene Gene used to alter bacteria for cleaning up toxic waste Gene for pest resistance inserted into plants Protein dissolvesblood clots in heartattack therapy Human growth hor-mone treats stuntedgrowth

  9. Fig. 20-2a Cell containing geneof interest Bacterium 1 Gene inserted intoplasmid Bacterialchromosome Plasmid Gene ofinterest RecombinantDNA (plasmid) DNA of chromosome 2 2 Plasmid put intobacterial cell Recombinantbacterium

  10. Fig. 20-2b Recombinantbacterium Host cell grown in cultureto form a clone of cellscontaining the “cloned”gene of interest 3 Protein expressedby gene of interest Gene ofInterest Copies of gene Protein harvested Basic research andvarious applications 4 Basicresearchon protein Basicresearchon gene Protein dissolvesblood clots in heartattack therapy Human growth hor-mone treats stuntedgrowth Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste

  11. Using Restriction Enzymes to Make Recombinant DNA • Bacterial restriction enzymes cut DNA molecules at specific 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 Animation: Restriction Enzymes

  12. DNA ligase is an enzyme that seals the bonds between restriction fragments

  13. Fig. 20-3-1 Restriction site 5 3 3 5 DNA Restriction enzymecuts sugar-phosphatebackbones. 1 Sticky end

  14. Fig. 20-3-2 Restriction site 5 3 3 5 DNA Restriction enzymecuts sugar-phosphatebackbones. 1 Sticky end DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs. 2 One possible combination

  15. Fig. 20-3-3 Restriction site 5 3 3 5 DNA Restriction enzymecuts sugar-phosphatebackbones. 1 Sticky end DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs. 2 One possible combination DNA ligaseseals strands. 3 Recombinant DNA molecule

  16. 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 host cell and replicate there

  17. Producing Clones of Cells Carrying Recombinant Plasmids • Several steps are required to clone the hummingbird β-globin gene in a bacterial plasmid: • The hummingbird genomic DNA and a bacterial plasmid are isolated • Both are digested with the same restriction enzyme • The fragments are mixed, and DNA ligase is added to bond the fragment sticky ends Animation: Cloning a Gene

  18. Some recombinant plasmids now contain hummingbird DNA • The DNA mixture is added to bacteria that have been genetically engineered to accept it • The bacteria are plated on a type of agar that selects for the bacteria with recombinant plasmids • This results in the cloning of many hummingbird DNA fragments, including the β-globin gene

  19. Fig. 20-4-1 Hummingbird cell TECHNIQUE Bacterial cell lacZ gene Restrictionsite Stickyends Gene of interest Bacterial plasmid ampR gene Hummingbird DNA fragments

  20. Fig. 20-4-2 Hummingbird cell TECHNIQUE Bacterial cell lacZ gene Restrictionsite Stickyends Gene of interest Bacterial plasmid ampR gene Hummingbird DNA fragments Nonrecombinant plasmid Recombinant plasmids

  21. Fig. 20-4-3 Hummingbird cell TECHNIQUE Bacterial cell lacZ gene Restrictionsite Stickyends Gene of interest Bacterial plasmid ampR gene Hummingbird DNA fragments Nonrecombinant plasmid Recombinant plasmids Bacteria carryingplasmids

  22. Fig. 20-4-4 Hummingbird cell TECHNIQUE Bacterial cell lacZ gene Restrictionsite Stickyends Gene of interest Bacterial plasmid ampR gene Hummingbird DNA fragments Nonrecombinant plasmid Recombinant plasmids Bacteria carryingplasmids RESULTS Colony carrying recombinant plasmid with disrupted lacZ gene Colony carrying non-recombinant plasmidwith intact lacZ gene One of manybacterial clones

  23. 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

  24. 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

  25. Fig. 20-6-1 DNA innucleus mRNAs in cytoplasm

  26. Fig. 20-6-2 DNA innucleus mRNAs in cytoplasm Reversetranscriptase Poly-A tail mRNA Primer DNAstrand

  27. Fig. 20-6-3 DNA innucleus mRNAs in cytoplasm Reversetranscriptase Poly-A tail mRNA Primer DNAstrand DegradedmRNA

  28. Fig. 20-6-4 DNA innucleus mRNAs in cytoplasm Reversetranscriptase Poly-A tail mRNA Primer DNAstrand DegradedmRNA DNA polymerase

  29. Fig. 20-6-5 DNA innucleus mRNAs in cytoplasm Reversetranscriptase Poly-A tail mRNA Primer DNAstrand DegradedmRNA DNA polymerase cDNA

  30. Expressing Cloned Eukaryotic Genes • After a gene has been cloned, its protein product can be produced in larger amounts for research • Cloned genes can be expressed as protein in either bacterial or eukaryotic cells

  31. 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

  32. 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

  33. Fig. 20-8 3 5 TECHNIQUE Targetsequence 3 5 Genomic DNA 1 5 3 Denaturation 5 3 2 Annealing Cycle 1yields 2 molecules Primers 3 Extension Newnucleo-tides Cycle 2yields 4 molecules Cycle 3yields 8 molecules;2 molecules(in whiteboxes)match targetsequence

  34. Fig. 20-8a 5 3 TECHNIQUE Targetsequence Genomic DNA 3 5

  35. Fig. 20-8b 1 5 3 Denaturation 3 5 2 Annealing Cycle 1yields 2 molecules Primers 3 Extension Newnucleo-tides

  36. Fig. 20-8c Cycle 2yields 4 molecules

  37. Fig. 20-8d Cycle 3yields 8 molecules;2 molecules(in whiteboxes)match targetsequence

  38. Concept 20.2: DNA technology allows us to study the sequence, expression, and function of a gene • DNA cloning allows researchers to • Compare genes and alleles between individuals • Locate gene expression in a body • Determine the role of a gene in an organism • Several techniques are used to analyze the DNA of genes

  39. Gel Electrophoresis and Southern Blotting • One indirect method of rapidly analyzing and comparing genomes is gel electrophoresis • This technique uses a gel as a molecular sieve to separate nucleic acids or proteins by size • A current is applied that causes charged molecules to move through the gel • Molecules are sorted into “bands” by their size Video: Biotechnology Lab

  40. Fig. 20-9 TECHNIQUE Powersource Mixture ofDNA mol-ecules ofdifferentsizes – Cathode Anode + Gel 1 Powersource – + Longermolecules 2 Shortermolecules RESULTS

  41. Fig. 20-9a TECHNIQUE Powersource Mixture ofDNA mol-ecules ofdifferentsizes Anode Cathode – + Gel 1 Powersource – + Longermolecules 2 Shortermolecules

  42. Fig. 20-9b RESULTS

  43. In restriction fragment analysis, DNA fragments produced by restriction enzyme digestion of a DNA molecule are sorted by gel electrophoresis • Restriction fragment analysis is useful for comparing two different DNA molecules, such as two alleles for a gene • The procedure is also used to prepare pure samples of individual fragments

  44. Fig. 20-10 Normal -globin allele Normalallele Sickle-cellallele 175 bp Large fragment 201 bp DdeI DdeI DdeI DdeI Largefragment Sickle-cell mutant -globin allele 376 bp 201 bp175 bp Large fragment 376 bp DdeI DdeI DdeI (a) DdeI restriction sites in normal and sickle-cell alleles of -globin gene (b) Electrophoresis of restriction fragments from normal and sickle-cell alleles

  45. A technique called Southern blotting combines gel electrophoresis of DNA fragments with nucleic acid hybridization • Specific DNA fragments can be identified by Southern blotting, using labeled probes that hybridize to the DNA immobilized on a “blot” of gel

  46. Fig. 20-11 TECHNIQUE Heavyweight Restrictionfragments I II III Nitrocellulosemembrane (blot) DNA + restriction enzyme Gel Sponge I Normal-globinallele II Sickle-cellallele III Heterozygote Papertowels Alkalinesolution 2 1 3 Preparation of restriction fragments DNA transfer (blotting) Gel electrophoresis Radioactively labeledprobe for -globin gene Probe base-pairswith fragments I II III I II III Fragment fromsickle-cell-globin allele Film overblot Fragment fromnormal -globin allele Nitrocellulose blot 4 5 Probe detection Hybridization with radioactive probe

  47. Fig. 20-11a TECHNIQUE Heavyweight Restrictionfragments I II III DNA + restriction enzyme Nitrocellulosemembrane (blot) Gel Sponge I Normal-globinallele II Sickle-cellallele III Heterozygote Papertowels Alkalinesolution 2 3 1 Preparation of restriction fragments Gel electrophoresis DNA transfer (blotting)

  48. Fig. 20-11b Radioactively labeledprobe for -globin gene Probe base-pairswith fragments I II III I II III Fragment fromsickle-cell-globin allele Film overblot Fragment fromnormal -globin allele Nitrocellulose blot 5 4 Hybridization with radioactive probe Probe detection

  49. Concept 20.3: Cloning organisms may lead to production of stem cells for research and other applications • Organismal cloning produces one or more organisms genetically identical to the “parent” that donated the single cell

  50. Cloning Plants: Single-Cell Cultures • One experimental approach for testing genomic equivalence is to see whether a differentiated cell can generate a whole organism • A totipotent cell is one that can generate a complete new organism

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