Dna Technology
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Chapter 20
DNA Technology and Genomics The following slides are for bonus… PowerPoint Lectures for Biology, Seventh Edition Neil Campbell and Jane Reece
Lectures by Chris Romero Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
• Overview: Understanding and Manipulating Genomes • One of the greatest achievements of modern science – Has been the sequencing of the human genome, which was largely complete by 2003
• DNA sequencing accomplishments – Have all depended on advances in DNA technology, starting with the invention of methods for making recombinant DNA Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
• 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 have developed methods for preparing well-defined, gene-sized pieces of DNA in multiple identical copies, a process called gene cloning
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
DNA Cloning and Its Applications: A Preview • Most methods for cloning pieces of DNA in the laboratory – Share certain general features, such as the use of bacteria and their plasmids
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
• Overview of gene cloning with a bacterial plasmid, showing various uses of cloned genes Bacterium
Cell containing gene 1 Gene inserted of interest into plasmid
Plasmid Bacterial chromosome Recombinant DNA (plasmid)
Gene of interest 2 Plasmid put into bacterial cell
Recombinate bacterium
3 Host cell grown in culture, to form a clone of cells containing the “cloned” gene of interest
Gene of interest
Protein expressed by gene of interest Protein harvested
Copies of gene
Universality of the genetic code enables this progress Figure 20.2
Basic research on gene
DNA of chromosome
4 Basic research and various applications
Basic research on protein
Gene used to alter Gene for pest Human growth Protein dissolves resistance inserted bacteria for cleaning blood clots in heart hormone treats up toxic waste stunted growth into plants attack therapy
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Using Restriction Enzymes to Make Recombinant DNA
• Bacterial restriction enzymes – Cut DNA molecules at a limited number of specific DNA sequences, called restriction sites
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
• A restriction enzyme will usually make many cuts in a DNA molecule – Yielding a set of restriction fragments
• The most useful restriction enzymes cut DNA in a staggered way – Producing fragments with “sticky ends” that can bond with complementary “sticky ends” of other fragments
• DNA ligase is an enzyme – That seals the bonds between restriction fragments Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
• Using a restriction enzyme and DNA ligase to make recombinant DNA Restriction site DNA 5′ 3′
3′ 5′
GAATTC CTTAAG
1 Restriction enzyme cuts
the sugar-phosphate backbones at each arrow AATTC
G CTTAA
G
Sticky end AATTC
2 DNA fragment from
another source is added. Base pairing of sticky ends produces various combinations. G AATT C C TTAA G
G
Fragment from different DNA molecule cut by the same restriction enzyme G AATTC CTTAA G
One possible combination 3 DNA ligase
seals the strands.
Figure 20.3
Recombinant DNA molecule
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
G CTTAA
Cloning a Eukraryotic Gene in a Bacterial Plasmid • In gene cloning, the original plasmid is called a cloning vector – Defined as a DNA molecule that can carry foreign DNA into a cell and replicate there
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Producing Clones of Cells APPLICATION Cloning is used to prepare many copies of a gene of interest for use in sequencing the gene, in producing its encoded protein, in gene therapy, or in basic research. TECHNIQUE
In this example, a human gene is inserted into a plasmid from E. coli. The plasmid contains the ampR gene, which makes E. coli cells resistant to the antibiotic ampicillin. It also contains the lacZ gene, which encodes β-galactosidase. This enzyme hydrolyzes a molecular mimic of lactose (X-gal) to form a blue product. Only three plasmids and three human DNA fragments are shown, but millions of copies of the plasmid and a mixture of millions of different human DNA fragments would be present in the samples.
1 Isolate plasmid DNA and human DNA.
2 Cut both DNA samples with the same restriction enzyme
3 Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids.
Figure 20.4 Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
lacZ gene (lactose Human breakdown) cell
Bacterial cell
Restriction site ampR gene (ampicillin resistance)
Bacterial plasmid
Gene of interest Sticky ends
Recombinant DNA plasmids
Human DNA fragments
• Concept 20.5: The practical applications of DNA technology affect our lives in many ways • Numerous fields are benefiting from DNA technology and genetic engineering
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Medical Applications • One obvious benefit of DNA technology – Is the identification of human genes whose mutation plays a role in genetic diseases
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Diagnosis of Diseases • Medical scientists can now diagnose hundreds of human genetic disorders – By using PCR and primers corresponding to cloned disease genes, then sequencing the amplified product to look for the diseasecausing mutation
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Human Gene Therapy • Gene therapy – Is the alteration of an afflicted individual’s genes – Holds great potential for treating disorders traceable to a single defective gene – Uses various vectors for delivery of genes into cells
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
• Gene therapy using a retroviral vector Cloned gene (normal allele, absent from patient’s cells)
Retrovirus capsid
1 Insert RNA version of normal allele into retrovirus. Viral RNA 2 Let retrovirus infect bone marrow cells that have been removed from the patient and cultured.
3 Viral DNA carrying the normal allele inserts into chromosome. Bone marrow cell from patient
Figure 20.16
4 Inject engineered cells into patient.
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Pharmaceutical Products • Applications of DNA technology include – Large-scale production of human hormones and other proteins with therapeutic uses – Production of safer vaccines
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Forensic Evidence • DNA “fingerprints” obtained by analysis of tissue or body fluids found at crime scenes – Can provide definitive evidence that a suspect is guilty or not – Can a woman leave DNA evidence at a crime scene?
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Environmental Cleanup • Genetic engineering can be used to modify the metabolism of microorganisms – So that they can be used to extract minerals from the environment or degrade various types of potentially toxic waste materials
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Agricultural Applications • DNA technology – Is being used to improve agricultural productivity and food quality
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Animal Husbandry and “Pharm” Animals • Transgenic animals – Contain genes from other organisms
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
– Have been engineered to be pharmaceutical “factories”
Figure 20.18 Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Genetic Engineering in Plants • Agricultural scientists – Have already endowed a number of crop plants with genes for desirable traits
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
DNA Technology and Genomics The following slides are for bonus… PowerPoint Lectures for Biology, Seventh Edition Neil Campbell and Jane Reece
Lectures by Chris Romero Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
• Overview: Understanding and Manipulating Genomes • One of the greatest achievements of modern science – Has been the sequencing of the human genome, which was largely complete by 2003
• DNA sequencing accomplishments – Have all depended on advances in DNA technology, starting with the invention of methods for making recombinant DNA Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
• 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 have developed methods for preparing well-defined, gene-sized pieces of DNA in multiple identical copies, a process called gene cloning
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
DNA Cloning and Its Applications: A Preview • Most methods for cloning pieces of DNA in the laboratory – Share certain general features, such as the use of bacteria and their plasmids
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
• Overview of gene cloning with a bacterial plasmid, showing various uses of cloned genes Bacterium
Cell containing gene 1 Gene inserted of interest into plasmid
Plasmid Bacterial chromosome Recombinant DNA (plasmid)
Gene of interest 2 Plasmid put into bacterial cell
Recombinate bacterium
3 Host cell grown in culture, to form a clone of cells containing the “cloned” gene of interest
Gene of interest
Protein expressed by gene of interest Protein harvested
Copies of gene
Universality of the genetic code enables this progress Figure 20.2
Basic research on gene
DNA of chromosome
4 Basic research and various applications
Basic research on protein
Gene used to alter Gene for pest Human growth Protein dissolves resistance inserted bacteria for cleaning blood clots in heart hormone treats up toxic waste stunted growth into plants attack therapy
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Using Restriction Enzymes to Make Recombinant DNA
• Bacterial restriction enzymes – Cut DNA molecules at a limited number of specific DNA sequences, called restriction sites
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
• A restriction enzyme will usually make many cuts in a DNA molecule – Yielding a set of restriction fragments
• The most useful restriction enzymes cut DNA in a staggered way – Producing fragments with “sticky ends” that can bond with complementary “sticky ends” of other fragments
• DNA ligase is an enzyme – That seals the bonds between restriction fragments Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
• Using a restriction enzyme and DNA ligase to make recombinant DNA Restriction site DNA 5′ 3′
3′ 5′
GAATTC CTTAAG
1 Restriction enzyme cuts
the sugar-phosphate backbones at each arrow AATTC
G CTTAA
G
Sticky end AATTC
2 DNA fragment from
another source is added. Base pairing of sticky ends produces various combinations. G AATT C C TTAA G
G
Fragment from different DNA molecule cut by the same restriction enzyme G AATTC CTTAA G
One possible combination 3 DNA ligase
seals the strands.
Figure 20.3
Recombinant DNA molecule
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
G CTTAA
Cloning a Eukraryotic Gene in a Bacterial Plasmid • In gene cloning, the original plasmid is called a cloning vector – Defined as a DNA molecule that can carry foreign DNA into a cell and replicate there
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Producing Clones of Cells APPLICATION Cloning is used to prepare many copies of a gene of interest for use in sequencing the gene, in producing its encoded protein, in gene therapy, or in basic research. TECHNIQUE
In this example, a human gene is inserted into a plasmid from E. coli. The plasmid contains the ampR gene, which makes E. coli cells resistant to the antibiotic ampicillin. It also contains the lacZ gene, which encodes β-galactosidase. This enzyme hydrolyzes a molecular mimic of lactose (X-gal) to form a blue product. Only three plasmids and three human DNA fragments are shown, but millions of copies of the plasmid and a mixture of millions of different human DNA fragments would be present in the samples.
1 Isolate plasmid DNA and human DNA.
2 Cut both DNA samples with the same restriction enzyme
3 Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids.
Figure 20.4 Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
lacZ gene (lactose Human breakdown) cell
Bacterial cell
Restriction site ampR gene (ampicillin resistance)
Bacterial plasmid
Gene of interest Sticky ends
Recombinant DNA plasmids
Human DNA fragments
• Concept 20.5: The practical applications of DNA technology affect our lives in many ways • Numerous fields are benefiting from DNA technology and genetic engineering
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Medical Applications • One obvious benefit of DNA technology – Is the identification of human genes whose mutation plays a role in genetic diseases
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Diagnosis of Diseases • Medical scientists can now diagnose hundreds of human genetic disorders – By using PCR and primers corresponding to cloned disease genes, then sequencing the amplified product to look for the diseasecausing mutation
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Human Gene Therapy • Gene therapy – Is the alteration of an afflicted individual’s genes – Holds great potential for treating disorders traceable to a single defective gene – Uses various vectors for delivery of genes into cells
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
• Gene therapy using a retroviral vector Cloned gene (normal allele, absent from patient’s cells)
Retrovirus capsid
1 Insert RNA version of normal allele into retrovirus. Viral RNA 2 Let retrovirus infect bone marrow cells that have been removed from the patient and cultured.
3 Viral DNA carrying the normal allele inserts into chromosome. Bone marrow cell from patient
Figure 20.16
4 Inject engineered cells into patient.
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Pharmaceutical Products • Applications of DNA technology include – Large-scale production of human hormones and other proteins with therapeutic uses – Production of safer vaccines
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Forensic Evidence • DNA “fingerprints” obtained by analysis of tissue or body fluids found at crime scenes – Can provide definitive evidence that a suspect is guilty or not – Can a woman leave DNA evidence at a crime scene?
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Environmental Cleanup • Genetic engineering can be used to modify the metabolism of microorganisms – So that they can be used to extract minerals from the environment or degrade various types of potentially toxic waste materials
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Agricultural Applications • DNA technology – Is being used to improve agricultural productivity and food quality
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Animal Husbandry and “Pharm” Animals • Transgenic animals – Contain genes from other organisms
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
– Have been engineered to be pharmaceutical “factories”
Figure 20.18 Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Genetic Engineering in Plants • Agricultural scientists – Have already endowed a number of crop plants with genes for desirable traits
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
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