Techniques of Recombinant DNA Technology

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Copyright © 2011 Pearson Education Inc.

Lecture prepared by Mindy Miller
-
Kittrell,
University of Tennessee, Knoxville

M I C R O B I O L O G Y

WITH DISEASES BY TAXONOMY, THIRD EDITION

Chapter 8

Recombinant DNA Technology

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The Role of Recombinant DNA technology 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 technology

Figure 8.1

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The Tools of Recombinant DNA Technology


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 Tools of Recombinant DNA Technology


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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The Tools of Recombinant DNA Technology


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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The Tools of Recombinant DNA Technology


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 enzymes

Figure 8.2

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The Tools of Recombinant DNA Technology

Animation: Recombinant DNA Technology

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The Tools of Recombinant DNA Technology


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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Producing a recombinant vector

Figure 8.3

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The Tools of Recombinant DNA Technology


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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Production of a gene library

Figure 8.4

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Techniques of Recombinant DNA Technology


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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Techniques of Recombinant DNA Technology


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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Techniques of Recombinant DNA Technology

Animation: Polymerase Chain Reaction: Overview

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Techniques of Recombinant DNA Technology

Animation: Polymerase Chain Reaction: Components

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Polymerase chain reaction (PCR)

Figure 8.5a

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Polymerase chain reaction (PCR)

Figure 8.5b

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Techniques of Recombinant DNA Technology

Animation: PCR: The Process

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Techniques of Recombinant DNA Technology


Selecting a Clone of Recombinant Cells


Must find clone containing DNA of interest


Probes are used

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Techniques of Recombinant DNA Technology


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 electrophoresis

Figure 8.6

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Techniques of Recombinant DNA Technology


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 technique

Figure 8.7

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Techniques of Recombinant DNA Technology


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 microarray

Figure 8.8

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Techniques of Recombinant DNA Technology


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 cells

Figure 8.9a/b

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Artificial methods of inserting DNA into cells

Figure 8.9c/d

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Applications of Recombinant DNA Technology


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 hybridization

Figure 8.10

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Automated DNA sequencing

Figure 8.11

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Applications of Recombinant DNA Technology


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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Applications of Recombinant DNA Technology


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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Applications of Recombinant DNA Technology


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 fingerprinting

Figure 8.12

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Applications of Recombinant DNA Technology


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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Applications of Recombinant DNA Technology


Agricultural Applications


Production of transgenic organisms


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



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Applications of Recombinant DNA Technology


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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Applications of Recombinant DNA Technology


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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Applications of Recombinant DNA Technology


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 DNA 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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The Ethics and Safety of Recombinant DNA Technology


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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The Ethics and Safety of Recombinant DNA Technology


Ethical Issues


Routine screenings?


Who should pay?


Genetic privacy rights?


Profits from genetically altered organisms?


Required genetic screening?


Forced correction of “genetic abnormalities”?