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16
Recombinant DNA and
Biotechnology
16 Recombinant DNA and Biotechnology
• 16.1 How Are Large DNA Molecules Analyzed?
• 16.2 What Is Recombinant DNA?
• 16.3 How Are New Genes Inserted into Cells?
• 16.4 What Are the Sources of DNA Used in
Cloning?
• 16.5 What Other Tools Are Used to Manipulate
DNA?
• 16.6 What Is Biotechnology?
16.1 How Are Large DNA Molecules Analyzed?
Naturally occurring enzymes that cleave
and repair DNA are used in the
laboratory to manipulate and recombine
DNA.
16.1 How Are Large DNA Molecules Analyzed?
Restriction enzymes (restriction
endonucleases) cut double-stranded
DNA into smaller pieces.
Bacteria use these as defense against
DNA from bacteriophage.
DNA is cut between the 3′ hydroxyl group
of one nucleotide and the 5′ phosphate
group of the next—restriction
digestion.
Figure 16.1 Bacteria Fight Invading Viruses with Restriction Enzymes
16.1 How Are Large DNA Molecules Analyzed?
There are many restriction enzymes that
cut DNA at specific base sequences—
the recognition sequence, or
restriction site.
16.1 How Are Large DNA Molecules Analyzed?
Restriction enzymes do not cut bacteria’s
own DNA because the recognition
sequences are modified.
Methylases add methyl groups after
replication; makes sequence
unrecognizable by restriction enzyme.
16.1 How Are Large DNA Molecules Analyzed?
Bacterial restriction enzymes can be
isolated from cells.
DNA from any organism will be cut
wherever the recognition site occurs.
EcoRI (from E. coli) cuts DNA at this
sequence:
16.1 How Are Large DNA Molecules Analyzed?
The sequence is palindromic—it reads
the same in both directions from the 5′
end.
EcoRI occurs about once every four
genes in prokaryotes. DNA can be
chopped into small pieces containing a
few genes.
16.1 How Are Large DNA Molecules Analyzed?
The EcoRI sequence does not occur
anywhere in the genome of the phage
T7. Thus it can survive in its host, E.
coli.
16.1 How Are Large DNA Molecules Analyzed?
After DNA is cut, fragments of different
sizes can be separated by gel
electrophoresis.
Mixture of fragments is place on a well in
a porous gel. An electric field is applied
across the gel. Negatively charged DNA
fragments move towards positive end.
Smaller fragments move faster than
larger ones.
Figure 16.2 Separating Fragments of DNA by Gel Electrophoresis (Part 1)
Figure 16.2 Separating Fragments of DNA by Gel Electrophoresis (Part 2)
Figure 16.2 Separating Fragments of DNA by Gel Electrophoresis (Part 3)
16.1 How Are Large DNA Molecules Analyzed?
Electrophoresis provides information on:
• Size of fragments. Fragments of known
size provide comparison.
• Presence of specific sequences. These
can be determined using probes.
DNA is denatured while in the gel, then
transferred to a nylon filter to make a
“blot.”
Figure 16.3 Analyzing DNA Fragments by Southern Blotting
16.1 How Are Large DNA Molecules Analyzed?
DNA fingerprinting uses restriction
analysis and electrophoresis to identify
individuals.
Works best with genes that are
polymorphic—have multiple alleles.
16.1 How Are Large DNA Molecules Analyzed?
Two types of polymorphisms:
Single nucleotide polymorphisms
(SNPs): inherited variation involving a
single base
Short tandem repeats (STRs):
moderately repetitive sequences side by
side
16.1 How Are Large DNA Molecules Analyzed?
STRs are recognizable if they lie
between two restriction sites.
Several different STRs can be used to
determine the unique pattern for an
individual.
Figure 16.4 DNA Fingerprinting with Short Tandem Repeats
16.1 How Are Large DNA Molecules Analyzed?
DNA fingerprinting requires at least 1 μg
of DNA (amount in about 100,000
human cells).
This is not always available, so
amplification by PCR is used.
16.1 How Are Large DNA Molecules Analyzed?
DNA fingerprinting is used in forensics.
It is more often used to prove innocence
than guilt.
Only a small portion of the genome is
examined; there is the possibility that
two people could have the same
sequence.
16.1 How Are Large DNA Molecules Analyzed?
DNA fingerprinting has been used to
analyze historical events.
The skeletal remains of Russian Tsar
Nicholas II and his family were identified
from DNA in bone fragments.
DNA also showed relationships with living
descendents of the Tsar.
Figure 16.5 DNA Fingerprinting the Russian Royal Family
16.1 How Are Large DNA Molecules Analyzed?
DNA technology can be used to identify
species.
A proposal to identify all known species
and look for unknowns has been put
forth by the Consortium for the Barcode
of Life (CBOL):
Use a short sequence from a gene
(cytochrome oxidase) as a “barcode” for
each species.
Figure 16.6 A DNA Barcode
16.1 How Are Large DNA Molecules Analyzed?
The barcode project could contribute to:
• Evolution research
• Species diversity issues
• Identification of new species
• Identification of undesirable microbes or
bioterrorism agents
16.2 What Is Recombinant DNA?
DNA fragments can be rejoined by DNA
ligase.
Any two DNA sequences can be spliced.
First done in 1973 with two E. coli
plasmids; Recombinant DNA was born
Figure 16.7 Making Recombinant DNA (Part 1)
Figure 16.7 Making Recombinant DNA (Part 2)
16.2 What Is Recombinant DNA?
Some restriction enzymes cut both DNA
strands exactly opposite each other.
Others (such as EcoRI) make a staggered
cut. Results in single-stranded “tails” at
the ends of fragments.
Tails are called sticky ends—can bind by
base pairing to other sticky ends.
Figure 16.8 Cutting and Splicing DNA
16.2 What Is Recombinant DNA?
Sticky ends of fragments that were cut by
the same restriction enzyme are all the
same—thus fragments from different
species can be joined.
When temperature is lowered, the
fragments anneal—join by hydrogen
bonding. Must be permanently spliced
by DNA ligase.
16.3 How Are New Genes Inserted into Cells?
Recombinant DNA technology can be
used to clone, or make exact copies of
genes.
The gene can be used to make a
protein—but it must first be inserted, or
transfected, into host cells.
The altered host cell is called
transgenic.
16.3 How Are New Genes Inserted into Cells?
To determine which of the host cells
contain the new sequence, the
recombinant DNA is often tagged with
reporter genes.
Reporter genes have easily observed
phenotypes or genetic markers.
16.3 How Are New Genes Inserted into Cells?
The first host cells used were bacteria,
especially E. coli.
Yeasts (Saccharomyces) are commonly
used as eukaryotic hosts.
Plant cells are also used—they have
totipotency, the ability of any
differentiated cell to develop into a new
plant.
16.3 How Are New Genes Inserted into Cells?
The new DNA must also replicate as the
host cell divides. It must become a
segment with an origin of replication—a
replicon or replication unit.
16.3 How Are New Genes Inserted into Cells?
New DNA can become part of a replicon
in two ways:
Inserted near an origin of replication in
host chromosome.
It can be part of a carrier sequence or
vector that already has an origin of
replication.
16.3 How Are New Genes Inserted into Cells?
A vector should have four characteristics:
• Ability to replicate independently of the
host cell
• A recognition sequence for a restriction
enzyme
• A reporter gene
• Small size in comparison with host’s
chromosomes
16.3 How Are New Genes Inserted into Cells?
Plasmids have all these characteristics.
• Plasmids are small, many have only
one restriction site.
• Genes for antibiotic resistance can be
used as reporter genes.
• And they have an origin of replication
and can replicate independently.
Figure 16.9 Vectors for Carrying DNA into Cells (A)
16.3 How Are New Genes Inserted into Cells?
Plasmids can be used for genes of
10,000 bp or less. Most eukaryote
genes are larger than this.
Viruses can be used as vectors—e.g.,
bacteriophage. The genes that cause
host cell to lyse can be cut out and
replaced with other DNA.
16.3 How Are New Genes Inserted into Cells?
Bacterial plasmids don’t work for yeasts
because the origins of replication use
different sequences.
A yeast artificial chromosome (YAC)
has been created: contains yeast origin
of replication, plus yeast centromere
and telomere sequences.
Also contains artificial restriction sites
and reporter genes
Figure 16.9 Vectors for Carrying DNA into Cells (B)
16.3 How Are New Genes Inserted into Cells?
A plasmid from the soil bacterium
Agrobacterium tumefaciens is used as a
vector for plant cells.
Plasmid Ti (tumor inducing) causes
crown gall.
Plasmid has a region called T DNA,
which inserts copies of itself into
chromosomes of infected plants.
16.3 How Are New Genes Inserted into Cells?
T DNA has several restriction sites,
where new DNA can be inserted.
With altered T DNA, plasmid no longer
causes tumors, but can still insert itself
into host chromosomes.
Figure 16.9 Vectors for Carrying DNA into Cells (C)
16.3 How Are New Genes Inserted into Cells?
Usually only a small proportion of host
cells take up the vector, and they may
not have the appropriate sequence.
Host cells with the desired sequence
must be identifiable.
16.3 How Are New Genes Inserted into Cells?
One method:
E. coli is host; pBR322 plasmid is the
vector.
Plasmid has genes for resistance to
ampicillin and tetracycline.
Plasmid has only one restriction site for
enzyme BamHI, within the gene for
tetracycline resistance.
16.3 How Are New Genes Inserted into Cells?
If new DNA is inserted at that restriction
site, it inactivates the gene for
tetracycline resistance.
Plasmid then has gene for ampicillin
resistance, but not for tetracycline. This
can be used to select for host cells with
new DNA.
Figure 16.10 Marking Recombinant DNA by Inactivating a Gene
16.3 How Are New Genes Inserted into Cells?
Other reporter genes:
• Artificial vectors with restriction sites
within the lac operon. If new DNA is
inserted there, vector no longer carries
its original function into the host cell.
• Green fluorescent protein, which
normally occurs in the jellyfish
Aequopora victoriana.
16.4 What Are the Sources of DNA Used in Cloning?
DNA fragments used for cloning come
from three sources:
• Gene libraries
• Reverse transcription from mRNA
• Artificial synthesis or mutation of DNA
16.4 What Are the Sources of DNA Used in Cloning?
Human chromosomes contain an
average of 80 million bp each.
The DNA is cut into fragments by
restriction enzymes, the fragments are
“stored” as a gene library.
Each fragment is inserted into a vector,
which goes into a host cell.
Figure 16.11 Constructing a Gene Library
16.4 What Are the Sources of DNA Used in Cloning?
If phage λ is used as a vector, about
50,000 volumes are required to store
the library.
One petri plate can hold 80,000 phage
colonies, or plaques.
DNA in the plaques is screened using
specific probes.
16.4 What Are the Sources of DNA Used in Cloning?
Smaller DNA libraries can be made from
complementary DNA (cDNA).
mRNA is extracted from a tissue and the
poly A tails allowed to hybridize with
oligo dT—a string of thymine bases.
Oligo dT serves as a primer for reverse
transcriptase to synthesize a
complementary DNA strand.
Figure 16.12 Synthesizing Complementary DNA
16.4 What Are the Sources of DNA Used in Cloning?
cDNA libraries are made from particular
tissues at particular times and represent
a snapshot of the mRNA present at that
time.
Used to compare gene expression in
different tissues at different stages of
development.
cDNA is also used to clone eukaryotic
genes.
16.4 What Are the Sources of DNA Used in Cloning?
DNA can be synthesized if the amino
acid sequence of a protein is known.
This process is now automated, and labs
can make custom DNA sequences
overnight.
Flanking sequences for transcription
initiation, termination, and regulation
and start and stop codons are also
added.
16.4 What Are the Sources of DNA Used in Cloning?
Synthetic DNA can be used to create
specific mutations in order to study the
consequences of the mutation.
Called mutagenesis techniques.
These techniques have revealed many
cause-and-effect relationships, e.g.,
determining signal sequences.
16.5 What Other Tools Are Used to Manipulate DNA?
Three additional ways of manipulating
DNA:
• Knockout experiments
• Gene silencing
• DNA chips
16.5 What Other Tools Are Used to Manipulate DNA?
A knockout experiment involves
homologous replication to replace a
gene with an inactive gene, and
determine results in a living organism.
The normal allele of a gene is inserted
into a plasmid; restriction enzymes are
used to insert a reporter gene in the
middle of the normal gene.
16.5 What Other Tools Are Used to Manipulate DNA?
The gene is thus inactivated.
The plasmid is then transfected into a
stem cell of a mouse embryo.
Stem cell: undifferentiated cell that
divides and differentiates to form
different tissues.
16.5 What Other Tools Are Used to Manipulate DNA?
Much of the normal gene is still present,
so homologous recognition takes place
between the normal allele and the
inactive allele on the plasmid.
Recombination can occur, and inactive
allele is swapped for the normal allele.
The transfected stem cell is then inserted
into an early mouse embryo.
Figure 16.13 Making a Knockout Mouse (Part 1)
Figure 16.13 Making a Knockout Mouse (Part 2)
16.5 What Other Tools Are Used to Manipulate DNA?
Translation of mRNA can be blocked by
complementary micro RNAs—
antisense RNA.
Antisense RNA can be synthesized, and
added to cells to prevent translation—
the effects of the missing protein can
then be determined.
16.5 What Other Tools Are Used to Manipulate DNA?
Interference RNA (RNAi) is a rare
natural mechanism that blocks
translation.
Short, double stranded RNA is unwound
and binds to complementary mRNA by
a protein complex, which also catalyzes
the breakdown of the mRNA.
Small interfering RNA (siRNA) can be
synthesized in the laboratory.
Figure 16.14 Using Antisense RNA and RNAi to Block Translation of mRNA
16.5 What Other Tools Are Used to Manipulate DNA?
Antisense RNA and RNAi are also used
to study cause-and-effect relationships.
Example: Antisense RNA is used to block
translation of proteins essential for
growth of cancer cells—the cells revert
to normal phenotype.
16.5 What Other Tools Are Used to Manipulate DNA?
DNA chip technology provides a large
array of sequences for hybridization
experiments.
A series of DNA sequences are attached
to a glass slide in a precise order.
The slide has microscopic wells which
each contain thousands of copies of
sequences up to 20 nucleotides long.
Figure 16.15 DNA on a Chip
16.5 What Other Tools Are Used to Manipulate DNA?
To analyze mRNA, it is incubated with
reverse transcriptase to make cDNA.
The cDNA is amplified using PCR.
Technique is called RT-PCR.
Amplified cDNA is tagged with a
fluorescent dye and used as a probe of
the DNA on the chip.
16.5 What Other Tools Are Used to Manipulate DNA?
DNA chip technology has been
developed to identify gene expression
patterns in women with a propensity for
breast cancer tumors to recur—a gene
expression signature.
16.6 What Is Biotechnology?
Biotechnology is the use of living cells to
produce materials useful to people.
Examples: use of yeasts to brew beer
and wine, use of bacteria to produce
cheese, yogurt, etc.
Use of microbes to produce antibiotics
such as penicillin, alcohol, and other
products.
16.6 What Is Biotechnology?
Gene cloning is now used to produce
proteins in large amounts.
Almost any gene can be inserted into
bacteria or yeasts, and the resulting
cells induced to make large quantities of
the product.
Requires specialized vectors.
16.6 What Is Biotechnology?
Expression vectors are synthesized
that include sequences needed for
expression of the transgene in the host
cell.
Figure 16.16 An Expression Vector Allows a Transgene to Be Expressed in a Host Cell
16.6 What Is Biotechnology?
Expression vectors can be modified by:
• Inducible promoters; enhancers can
also be added so that protein synthesis
takes place at high rates.
• Tissue-specific promoters
• Signal sequences—e.g., a signal to
secrete the product to the extracellular
medium.
Table 16.1
16.6 What Is Biotechnology?
Example of a medical application:
After wounds heal, blood clots are
dissolved by plasmin. Plasmin is stored
as an inactive form called plasminogen.
Conversion of plasminogen is activated by
tissue plasminogen activator (TPA).
TPA can be used to treat strokes and heart
attacks, but large quantities are needed—
can be made using recombinant DNA
technology.
Figure 16.17 Tissue Plasminogen Activator: From Protein to Gene to Drug (Part 1)
Figure 16.17 Tissue Plasminogen Activator: From Protein to Gene to Drug (Part 2)
16.6 What Is Biotechnology?
Pharming: production of medically useful
proteins in milk.
Transgenes for a protein are inserted into
the egg of a domestic animal, next to
the promoter for lactoglobulin—a protein
in milk. The transgenic animal then
produces large quantities of the protein
in its milk.
Figure 16.18 Pharming
16.6 What Is Biotechnology?
Through cultivation and selective breeding,
humans have been altering the traits of
plants and animals for thousands of years.
Recombinant DNA technology has several
advantages:
• Specific genes can be targeted.
• Any gene can be introduced into any other
organism.
• New organisms are generated quickly.
Table 16.2
16.6 What Is Biotechnology?
Crop plants have been modified to
produce their own insecticides:
• The bacterium Bacillus thuringiensis
produces a protein that kills insect
larvae.
• Dried preparation of B. thuringiensis are
sold as a safe alternative to synthetic
insecticides. The toxin is easily
biodegradable.
16.6 What Is Biotechnology?
Genes for the toxin have been isolated,
cloned, and modified, and inserted into
plant cells using the Ti plasmid vector.
Transgenic corn, cotton, soybeans,
tomatoes, and other crops are being
grown. Pesticide use is reduced.
16.6 What Is Biotechnology?
Some transgenic crops are resistant to
herbicides.
Glyphosate (Roundup) is widely used to
kill weeds.
Expression vectors have been used to
make plants that synthesize so much of
the target enzyme of glyphosate that
they are unaffected by the herbicide.
16.6 What Is Biotechnology?
The gene has been inserted into corn,
soybeans, and cotton.
About half of U.S. crops of these plants
contain this gene.
16.6 What Is Biotechnology?
Crops with improved nutritional
characteristics:
• Rice does not have β-carotene, but
does have a precursor molecule.
• Genes for enzymes that synthesize βcarotene from the precursor are taken
from daffodils and inserted into rice by
the Ti plasmid.
16.6 What Is Biotechnology?
• The transgenic rice is yellow, and can
supply β-carotene to improve the diets
of many people.
• β-carotene is converted to vitamin A in
the body.
Figure 16.19 Transgenic Rice Is Rich in β-Carotene
16.6 What Is Biotechnology?
Recombinant DNA is also used to adapt
a crop plant to an environment.
Example: plants that are salt-tolerant
Genes from a protein that moves sodium
ions into the central vacuole were
isolated from Arabidopsis and inserted
into tomato plants.
Figure 16.20 Salt-Tolerant Tomato Plants
16.6 What Is Biotechnology?
Concerns over biotechnology:
• Genetic manipulation is an unnatural
interference in nature.
• Genetically altered foods are unsafe to
eat.
• Genetically altered crop plants are
dangerous to the environment.
16.6 What Is Biotechnology?
Advocates of biotechnology point out that
all crop plants have been manipulated
by humans.
Advocates say that since only single
genes for plant function are inserted into
crop plants, they are still safe for human
consumption.
Genes that affect human nutrition may
raise more concerns.
16.6 What Is Biotechnology?
Concern over environmental effects
centers on escape of transgenes into
wild populations:
For example, if the gene for herbicide
resistance made its way into the weed
plants.
Beneficial insects can also be killed from
eating plants with B. thuringiensis
genes.