Recombinant DNA Technology 1. The Role of Recombinant DNA technology in Biotechnology Recombinant DNA technology ◦Intentionally modifying genomes of organisms

Recombinant DNA Recombinant DNA TechnologyTechnology

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The Role of Recombinant DNA The Role of Recombinant DNA technology in Biotechnologytechnology in Biotechnology

Recombinant DNA technology◦Intentionally modifying genomes of organisms

for practical purposes◦Three goals

Eliminate undesirable phenotypic traits Combine beneficial traits of two or more

organisms Create organisms that synthesize products

humans need

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Overview of recombinant DNA Overview of recombinant DNA technologytechnology

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Figure 8.1

The Tools of Recombinant DNA The Tools of Recombinant DNA TechnologyTechnology

Mutagens◦Physical and chemical agents that produce

mutations◦Scientists utilize mutagens to

◦Create changes in microbes’ genomes to change phenotypes

◦Select for and culture cells with beneficial characteristics

◦Mutated genes alone can be isolated

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The Use of Reverse Transcriptase to Synthesize cDNA◦Isolated from retroviruses◦Uses RNA template to transcribe molecule of

cDNA◦Easier to isolate mRNA molecule for desired

protein first◦mRNA of eukaryotes has introns removed

Allows cloning in prokaryotic cells

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Synthetic Nucleic Acids◦Molecules of DNA and RNA produced in cell-free

solutions◦Uses of synthetic nucleic acids

Elucidating the genetic code Creating genes for specific proteins Synthesizing DNA and RNA probes to locate

specific sequences of nucleotides Synthesizing antisense nucleic acid molecules

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Restriction Enzymes◦Bacterial enzymes that cut DNA molecules only

at restriction sites◦Categorized into two groups based on type of cut

Cuts with sticky ends Cuts with blunt ends

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Actions of restriction enzymesActions of restriction enzymes8

Figure 8.2


Vectors◦Nucleic acid molecules that deliver a gene into

a cell◦Useful properties

Small enough to manipulate in a lab Survive inside cells Contain recognizable genetic marker Ensure genetic expression of gene

◦Include viral genomes, transposons, and plasmids

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Gene Libraries◦A collection of bacterial or phage clones

Each clone in library often contains one gene of an organism’s genome

◦Library may contain all genes of a single chromosome

◦Library may contain set of cDNA complementary to mRNA

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Techniques of Recombinant DNA Techniques of Recombinant DNA TechnologyTechnology

Multiplying DNA in vitro: The Polymerase Chain Reaction (PCR)◦Large number of identical molecules of DNA

produced in vitro◦Critical to amplify DNA in variety of situations

Epidemiologists use to amplify genome of unknown pathogen

Amplified DNA from Bacillus anthracis spores in 2001 to identify source of spores

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Multiplying DNA in vitro: The Polymerase Chain Reaction (PCR)◦Repetitive process consisting of three steps

Denaturation Priming Extension

◦Can be automated using a thermocycler

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Polymerase chain reaction (PCR)Polymerase chain reaction (PCR)13

Figure 8.5a

Polymerase chain reaction (PCR)Polymerase chain reaction (PCR)14

Figure 8.5b


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Selecting a Clone of Recombinant Cells◦Must find clone containing DNA of interest◦Probes are used

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Separating DNA Molecules: Gel Electrophoresis and the Southern Blot◦Gel electrophoresis

Separates molecules based on electrical charge, size, and shape

Allows scientists to isolate DNA of interest Negatively charged DNA drawn toward positive

electrode Agarose makes up gel; acts as molecular sieve Smaller fragments migrate faster and farther than

larger ones Determine size by comparing distance migrated to

standards

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Gel electrophoresisGel electrophoresis18

Figure 8.6


Separating DNA Molecules: Gel Electrophoresis and the Southern Blot◦Southern blot

DNA transferred from gel to nitrocellulose membrane Probes used to localize DNA sequence of interest Northern blot – used to detect RNA

◦Uses of Southern blots Genetic “fingerprinting” Diagnosis of infectious disease Demonstrate incidence and prevalence of organisms

that cannot be cultured

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The Southern blot techniqueThe Southern blot technique20

Figure 8.7


DNA Microarrays◦Consist of molecules of immobilized single-

stranded DNA◦Fluorescently labeled DNA washed over array

will adhere only at locations where there are complementary DNA sequences

◦Variety of scientific uses of DNA microarrays Monitoring gene expression Diagnosis of infection Identification of organisms in an environmental

sample

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DNA microarrayDNA microarray22

Figure 8.8


Inserting DNA into Cells◦Goal of DNA technology is insertion of DNA into

cell ◦Natural methods

Transformation Transduction Conjugation

◦Artificial methods Electroporation Protoplast fusion Injection – gene gun and microinjection

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Artificial methods of inserting DNA into Artificial methods of inserting DNA into cellscells

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Figure 8.9a/b

Artificial methods of inserting DNA into Artificial methods of inserting DNA into cellscells

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Figure 8.9c/d

Applications of Recombinant DNA Applications of Recombinant DNA TechnologyTechnology

Genetic Mapping◦Locating genes on a nucleic acid molecule◦Provides useful facts concerning metabolism,

growth characteristics, and relatedness to others

Locating Genes◦Until 1970, genes identified by labor-intensive

methods◦Simpler and universal methods now available◦Restriction fragmentation◦Fluorescent in situ hybridization (FISH)

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Fluorescent in situ hybridizationFluorescent in situ hybridization27

Figure 8.10

Automated DNA sequencingAutomated DNA sequencing28

Figure 8.11


Environmental Studies◦Most microorganisms have never been grown in

a laboratory◦Scientists know them only by their DNA

fingerprints Allowed identification of over 500 species of

bacteria from human mouths Determined that methane-producing archaea are

a problem in rice agriculture

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Pharmaceutical and Therapeutic Applications◦Protein synthesis

Creation of synthetic peptides for cloning◦Vaccines

Production of safer vaccines Subunit vaccines Introduce genes of pathogens into common fruits

and vegetables Injecting humans with plasmid carrying gene

from pathogen◦Humans synthesize pathogen’s proteins

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Pharmaceutical and Therapeutic Applications◦Genetic screening

DNA microarrays used to screen individuals for inherited disease caused by mutations

Can also identify pathogen’s DNA in blood or tissues

◦DNA fingerprinting Identifying individuals or organisms by their

unique DNA sequence

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DNA fingerprintingDNA fingerprinting32

Figure 8.12


Pharmaceutical and Therapeutic Applications◦Gene therapy

Missing or defective genes replaced with normal copies

Some patients’ immune systems react negatively◦Medical diagnosis

Patient specimens can be examined for presence of gene sequences unique to certain pathogens

◦Xenotransplants Animal cells, tissues, or organs introduced into

human body

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Agricultural Applications◦Production of transgenic organisms

Recombinant plants and animals altered by addition of genes from other organisms

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Agricultural Applications◦Herbicide resistance

Gene from Salmonella conveys resistance to glyphosate (Roundup)◦Farmers can kill weeds without killing crops

◦Salt tolerance Scientists have removed gene for salt tolerance

and inserted into tomato and canola plants Transgenic plants survive, produce fruit, and

remove salt from soil

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Agricultural Applications◦Freeze resistance

Crops sprayed with genetically modified bacteria can tolerate mild freezes

◦Pest resistance Bt toxin

◦Naturally occurring toxin only harmful to insects ◦Organic farmers used to reduce insect damage to crops

Gene for Bt toxin inserted into various crop plants Genes for Phytophthora resistance inserted into

potato crops

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Agricultural Applications◦Improvements in nutritional value and yield

Tomatoes allowed to ripen on vine and shelf life increased◦Gene for enzyme that breaks down pectin suppressed

BGH allows cattle to gain weight more rapidly, ◦Have meat with lower fat content and produce 10%

more milk Gene for β-carotene (vitamin A precursor) inserted

into rice Scientists considering transplanting genes coding

for entire metabolic pathways

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The Ethics and Safety of Recombinant The Ethics and Safety of Recombinant DNA TechnologyDNA Technology

Supremacist view – humans are of greater value than animals

Long-term effects of transgenic manipulations are unknown

Unforeseen problems arise from every new technology and procedure

Natural genetic transfer could deliver genes from transgenic plants and animals into other organisms

Transgenic organisms could trigger allergies or cause harmless organisms to become pathogenic

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Studies have not shown any risks to human health or environment

Standards imposed on labs involved in recombinant DNA technology

Can create biological weapons using same technology

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Ethical Issues◦Routine screenings?◦Who should pay?◦Genetic privacy rights?◦Profits from genetically altered organisms?◦Required genetic screening?◦Forced correction of “genetic abnormalities”?

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Recombinant DNA Technology 1. The Role of Recombinant DNA technology in Biotechnology Recombinant DNA technology ◦Intentionally modifying genomes of organisms