Chapter 8

Recombinant
DNA
Technology

2012 Pearson Education Inc.

Lecture prepared by Mindy Miller-Kittrell

North Carolina State University

The Role of Recombinant DNA Technology in Biotechnology

Recombinant DNA Technology

Intentional modification of organisms genomes 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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Figure 8.1 Overview of recombinant DNA technology

Bacterial cell
DNA containing
gene of interest

Bacterial
chromosome

Plasmid
Isolate plasmid.

Gene of interest
Enzymatically cleave
DNA into fragments.

Isolate fragment
with the gene of
interest.

Insert gene into plasmid.

Insert plasmid and gene into

bacterium.

Culture bacteria.

Harvest copies of
gene to insert into
plants or animals

Eliminate
undesirable
phenotypic
traits

Create
beneficial
combination
of traits

Harvest proteins
coded by gene

Produce vaccines,
antibiotics,
hormones, or
enzymes

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 cellfree 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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Figure 8.2 Actions of restriction enzymes-overview

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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Figure 8.3 Producing a recombinant vector

mRNA for human

growth hormone (HGH)

Antibiotic
resistance
gene

Restriction
site
Reverse
transcription

cDNA for HGH

Plasmid (vector)
Restriction
enzyme

Restriction
enzyme

Sticky ends

Gene for human

growth hormone

Ligase

Recombinant plasmid
Introduce recombinant
plasmid into bacteria.

Bacterial
chromosome

Recombinant
plasmid
Inoculate bacteria
on media containing
antibiotic.
Bacteria containing
the plasmid with
HGH gene survive
because they also
have resistance gene.

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 organisms genome

Library may contain all genes of a single

chromosome
Library may contain set of cDNA
complementary to mRNA

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Figure 8.4 Production of a gene library-overview

Genome

Isolate genome
or organism.

Generate fragments using

restriction enzymes.

Insert each fragment

into a vector.

Introduce vectors
into cells.

Culture recombinant cells;

descendants are clones.

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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Figure 8.5a The use of PCR to replicate DNA, steps 1-3

Original DNA 3
molecule
5

3
5
Heat to 94C

Denaturation

DNA primer

Deoxyribonucleotide

Priming triphosphates

DNA polymerase

Cool to 65C
DNA polymerase
5

3
5
Extension

DNA primer
5
3

5
72C

Figure 8.5b The use of PCR to replicate DNA, step 4

First cycle

Second cycle

Third cycle

Fourth cycle

2 DNA
molecules
4 DNA
molecules

8 DNA
molecules

Repeat

16 DNA
molecules

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 than larger ones
Determine size by comparing distance migrated to
standards
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Figure 8.6 Gel electrophoresis-overview

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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Figure 8.7 The Southern blot technique-overview

DNA molecules
Restriction enzymes

Restriction fragments
Use gel electrophoresis to separate
fragments by size; denature DNA
into single strands with NaOH.
DNA

DNA bands
The DNA fragments
are invisible to the
investigators at
this stage.

Gel
Nitrocellulose
membrane
Absorbent
material

Side view

Electrophoresis
gel
Nitrocellulose
membrane
Absorbent
material

Nitrocellulose membrane
with DNA fragments at
same locations as in gel
(still invisible) is baked to
permanently affix DNA.

Add radioactive probes

complementary to DNA
nucleotide sequence
of interest.
Probes bind to DNA
of interest.

Incubate with film; radiation exposes film.

Develop film.
Developed film

Techniques of Recombinant DNA Technology

DNA Microarrays
Consist of molecules of immobilized singlestranded 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 of gene expression
Diagnosis of infection
Identification of organisms in an environmental
sample
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Figure 8.8 DNA microarray-overview

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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Figure 8.9a Artificial methods of inserting DNA into cells: electroporation

Pores in wall and membrane

Chromosome
Cell synthesizes
new wall
Electrical
field applied
Electroporation

Recombinant cell

Competent cell
DNA from
another source

Figure 8.9b Artificial methods of inserting DNA into cells: protoplast fusion

Cell synthesizes
new wall

Cell walls

Enzymes remove
cell walls

Polyethylene
glycol

Recombinant cell
New wall

Protoplasts
Protoplast fusion

Fused protoplasts

Figure 8.9c Artificial methods of inserting DNA into cells: gene gun

Blank .22
Nylon
caliber shell projectile

Vent

Plate to stop
nylon projectile

Target cell

DNA-coated beads

Protoplasts

Gene gun

Nylon
projectile

Figure 8.9d Artificial methods of inserting DNA into cells: microinjection

Micropipette
containing DNA

Target cells
nucleus
Target cell

Suction tube
to hold target
cell in place

Microinjection

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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Figure 8.10 FISH

Figure 8.11 Automated DNA sequencing

Nucleotide bases:
T

Signal intensity

100
Number of bases

200

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
Genes of pathogens introduced into common fruits
and vegetables
Injecting humans with plasmid carrying gene from
pathogen
Humans synthesize pathogens 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 pathogens DNA in blood or
tissues

DNA fingerprinting
Identifying individuals or organisms by their unique
DNA sequence

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Figure 8.12 DNA fingerprinting

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

Salt tolerance
Scientists have inserted gene for salt tolerance 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 harmful only 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

Produce meat with lower fat content and produce
10% more milk

Gene for -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?

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