Biotechnology and
Recombinant DNA
Chapter 9
Biotechnology and
Recombinant DNA
 Biotechnology

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The use of microbiological and biochemical
techniques to solve problems and produce
product
Recombinant DNA techniques

Methods used to manipulated DNA to
intentionally genetically alter organisms through
genetic engineering
 Often to give them more useful traits
Fundamental Tools of Biotechnology
 Basic components of molecular biologist’s
“toolkit”
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Restriction enzymes
Gel electrophoresis
DNA probes
Primers
Fundamental Tools of Biotechnology
 Restriction enzymes

Naturally occurring enzymes that cut DNA into
fragments


Cut in predictable and controllable manner
Generates pieces of DNA called restriction
fragments

These fragments can be joined to new fragments
 Enzymes produce jagged cuts called sticky ends
 Ends anneal together to form new strand

DNA ligase covalently joins fragments
Fundamental Tools of Biotechnology
 Gel electrophoresis

Used to separate DNA fragments according to
size

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DNA is put into wells in gel
Gel subjected to current
DNA moves through the gel
 Fragments are separated according to size
 Large fragments remain high in the gel
 Small fragments migrate lower

Gel must be stained to view DNA

Stained with ethidium bromide solution
Fundamental Tools of Biotechnology
 DNA probes


Used to locate nucleotide sequences in DNA
or RNA
Probe is single-stranded piece of DNA tagged
with detectable marker


Location can be easily determined
Probe will hybridize to complementary
fragment of interest
Fundamental Tools of Biotechnology
 Primers

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Single stranded DNA fragments the bind
sequences of DNA
Used in in vitro DNA synthesis

Primer serves fragment for addition of DNA
nucleotides
Applications of
Genetic Engineering
 Genetically engineered
bacteria

Genetic engineering
relies on DNA cloning
 Process of
producing copies of
DNA
 Cloned DNA
generally combined
with carrier molecule
called cloning vector
 Insures replication
of target DNA
Applications of
Genetic Engineering
 Genetically engineered organisms have
variety of uses



Protein production
DNA production
Researching gene function and regulation
Applications of
Genetic Engineering
 Protein production

Produce commercially important proteins

Pharmaceutical proteins
 Human insulin

Vaccines
 Hepatitis B vaccine

Commercially valuable proteins
 Chymosin used in the production of cheese
Applications of
Genetic Engineering
 DNA production


Researches interested in acquiring available
sources of specific DNA fragments
Fragments used for

DNA study
 Looking genomic characteristics

DNA vaccines
 Looking at injecting DNA of pathogen to produce
immune response
Applications of
Genetic Engineering
 Researching gene function and
regulation
 Function and regulation can
be more easily study in
certain bacteria


E. coli used often due to
established protocols
Gene expression can be
study by gene fusion

Joining gene being study to
reporter gene
 Reporter gene encodes
observable trait
 Trait makes it possible
to determine changes
in gene
Applications of
Genetic Engineering
 Genetically engineered eukaryotes
 Yeast serve as important eukaryotic model for gene
function and regulation
 Plant or animal that receive engineered gene termed
transgenic organism
 Examples of genetically altered plants include
 Pest resistant plants
 Corn, cotton and potatoes

Herbicide resistant plants
 Soybeans, cotton and corn

Plants with improved nutrient value
 Rice

Plants as edible vaccines
 Bananas and potatoes
Applications of
Probe Technologies
 Variety of technology employ DNA probes

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Colony blotting
Southern blotting
Fluorescence in situ hybridization (FISH)
DNA microarray
Applications of
Probe Technologies
 Colony blotting
 Used to detect specific DNA
sequences in colonies
grown in agar plates
 Colonies are transferred in
place on nylon membrane
 Colony blots are used to
determine which cell contain
gene of interest
Applications of
Probe Technologies
 Southern blotting
 Uses probes to detect
DNA sequences in
restriction fragments
separated using gel
electrophoresis
 Application of
Southern blotting is
locating DNA
sequences similar to
ones being studied
Applications of
Probe Technologies
 Fluorescence in situ hybridization (FISH)

Uses fluorescently labeled probed to detect
certain nucleotide sequences

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Detects sequences inside intact cell
Specimen is view using fluorescence
microscope
FISH can be used to identify specific
properties of bacteria

Mycobacterium tuberculosis in sputum sample
Applications of
Probe Technologies
 DNA microarray
technologies


DNA arrays are solid
supports with fixed patterns
of different single stranded
DNA fragments attached
Enables researches to
screen sample for
numerous sequences
simultaneously
Applications for
DNA Sequencing
 Knowing DNA sequence of particular cell
helps identify genetic alterations

Alterations that may result in disease

Sickle cell anemia
 Due to single base-pair change in gene

Cystic fibrosis
 Caused by three base-pair deletion
 DNA sequence analysis assists in studying
evolutionary relatedness
Applications of
Polymerase Chain Reaction
 Creates millions of
copies of given region
of DNA in matter of
hours

Technique exploits
specificity of primers
 Allows for selective
replication of chosen
regions
 Termed target DNA

Large amounts of
DNA can be
produced from very
small sample
 Care must be taken to
prevent contamination with
external source of target
DNA
 Basis for false-positive
test results
Techniques Used in
Genetic Engineering
 Obtaining DNA to be cloned
 Generally through cell lysis
 Generating a recombinant molecule
 Restriction enzymes and ligases used to create
recombinant molecule
 Introducing recombinant molecule into new host
 Host acts “incubator” for DNA replication
 DNA-mediated transformation often used to get
DNA into host
Techniques used in
Probe Technologies
 Probe technologies include


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Colony blotting
Southern blotting
FISH
Microarray technology
Techniques used in
Probe Technologies
 Techniques in colony and Southern blotting

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Blotting steps transfer sample to nylon
membrane
Probe is added

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Probe hybridizes with complementary sequence
Process is used to locate positions of
hybridized probe
Techniques used in
Probe Technologies
 Techniques used in FISH

Sample preparation is critical

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Specimen is applied to glass slide
Fluorescent label is applied and incubated

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Methods used depend on type of organism
Incubation allows for hybridization
Specimen is view with fluorescence
microscope
Techniques Used in
DNA Sequencing
 Dideoxychain termination
 Elements for termination reaction include

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Single-stranded DNA template
Primer that anneal to template
DNA polymerase
Each of the nucleotide bases
 One of these bases is labeled with marker for detection

Dideoxynucleotides
 Like deoxynucleotide counterparts but lack 3’ OH
 Incorporation causes chain termination

Special gel electrophoresis used to separate DNA
fragments by size
Techniques Used in
DNA Sequencing
 Automated DNA sequencing

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Most automated systems
use fluorescent dyes to
detect newly synthesized
DNA
Gel electrophoresis used
to separate fragments
into colored bands
Laser used to detect
color differences

Order of color reflects
nucleotide sequence
Techniques Used in
Polymerase Chain Reaction
 Starting with double stranded DNA molecule, process
involves number of amplification cycles
 PCR requires three step amplification cycle


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Step 1: double stranded DNA denatured by heat
Step 2: primers anneal to complementary sequence of
target DNA and DNA synthesis occurs with heat stable
DNA polymerase
Step 3: duplication of target DNA
 DNA is amplified exponentially