2ND

CYTOGENETICS YR

CHAPTER 19: DNA TECHNOLOGIES

OUTLINE  A patentable transgenic organism is the creation of

plants and animals with new useful traits by inserting
I Introduction one or more genes taken from other species.
II Treating cancer by location or mutation  A DNA sequence alone does not warrant patent
III Cancer: Abnormal growth that invades and spreads
protection. It must be useful as a tool for research or
IV Loss of Cycle Control
as a novel or improved product, such as a diagnostic
V Cancer at cellular Level
VI Cancer Genes and Genomes test or a drug.
VII Diagnostic and testing Cancer  A gene patent is the exclusive rights to a specific
sequence of DNA (a gene) given by a government to
the individual, organization, or corporation who
IMPROVING PIG MANURE claims to have first identified the gene.

 Pigs do not make an enzyme to extract the mineral

nutrient phosphorus from a compound called
phytate in cereal grains in their feed, so they are
given dietary phosphorus supplements which results
to phosphorous manure. [Pig manure]
 As pig raisers feed animal by-products, the
animals that consumed it can introduce
prion diseases
 Enviropig- a low-phosphorous manure
excreted by the animals due to secretion of
bacterial phytase in saliva of a genetically
modified “phytase transgenic pic”
o a transgenic
o has a phytase gene from the bacterium E.
coli
o has a promoter DNA sequence from a
mouse that controls secretion of phytase
from the salivary glands.
o has 75% less phosphorus than unaltered
pig excrement.
 Transgenic organism- has a gene in each of its cells
from an organism of another species.

PATERNING DNA
 Transgenic- Organisms that harbor DNA from other
species.
o their DNA is called recombinant
o Creating transgenic organisms is possible because
all life uses the same genetic code—that is, the
same DNA triplets encode the same amino acids.
o any other invention, must be new, useful, and not
obvious to an expert in the field
 DNA moves and mixes between species in nature
(eg., Bacteria DNA)
PATENTABLE
 DNA may be considered intellectual property and,
therefore, patentable.
 A DNA sequence might be patentable if it is part of a
medical device used to diagnose an inherited or
infectious disease.
MODIFYING DNA
 Recombinant DNA technology- adds genes from one
type of organism to the genome of another.
o first gene modification biotechnology—done in
bacteria to make them produce peptides and
proteins useful as drugs.
RECOMBINANT DNA
 Initially focused on providing direct gene products
such as peptides and proteins (eg., human versions
of insulin, growth hormone, and clotting factors).
 can target carbohydrates and lipids by affecting the
genes that encode enzymes required to synthesize
them.

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 The enzymes generate offset ends that are “sticky”
so that pieces connect by base-pairing.
 provides a way to isolate genes from complex
organisms and observe their functions on the
molecular level.
Constructing and Selecting Recombinant DNA molecules
 Manufacturing of recombinant DNA
molecules requires three components:
1. Restriction enzymes. Enzymes that cut
DNA at specific sequences (They are also
called restriction endonucleases because
they cut DNA within the molecule
[“endo”] rather than from the ends
[“exo”].)
 naturally found in bacteria, where
they cut DNA of infecting viruses,
protecting the bacteria.
 work as molecular scissors in
creating recombinant DNA
molecules
 Some restriction enzymes of a
bacteria cut DNA at particular
sequences of four, five, or six bases
that are symmetrical in a specific
way—the recognized sequence
reads the same, from the 5′ to 3′
direction, on both strands of the
DNA; this action creates single-
stranded extensions because the
individual cuts are slightly offset,
creating overhangs.
2. Cloning vectors. Pieces of DNA used to
deliver specific DNA sequences to cells
 carries DNA from the cells of one
species into the cells of another.
 Plasmid is a commonly used vector;
a small circle of double-stranded
DNA that exists naturally in some
bacteria, yeasts, plant cells, and cells
of other types of organisms.
 Bacteriophages another vector; a
virus that infect bacteria;
manipulated to transport DNA, but
not cause disease.
 Disabled retroviruses is also used as
vectores.
 Gene size must be short enough to
insert into the vector (kb—kilobases)
3. Recipient cells. bacteria or cultured
single cells
Insert DNA to vectors  loaded vectors are
delivered to selected cells  manufacture of
desired protein.
Selecting Recombinant DNA molecules
 distinguishing cells bearing the gene from
cells that lack plasmids or that have taken up
“empty” plasmids that do not contain the
gene.
 Separation strategy:
Use of plasmids that have an antibiotic resistance
gene as well as a gene that encodes an enzyme
that catalyzes a reaction that produces a blue
color. When the antibiotic is applied, only cells
that have plasmids survive.
o IF: a human gene inserts and interrupts
the gene for the enzyme, the bacterial
colony that grows is not blue and is,
therefore, easily distinguished from the
blue bacterial cells that have not taken
up the human gene.
Products from Recombinant DNA Technology
 Also used to mass-produce protein-based
drugs.
 Drugs manufactured using recombinant DNA
technology are pure, and are the human
version of the protein.
 Insulin- first drug manufactured using
recombinant DNA technology; produced in
bacterial cells (E. Coli)
o a simple peptide and is therefore
straightforward to mass-produce in
bacteria.
o must be produced in eukaryotic cells that
readily carry out these modifications.
 Cattle insulin- is the insulin used in patients
with type 1 DM; similar to the human
peptide, differing in only two of its 51 amino
acids, that most people with diabetes could
use it; about 20 patients is allergic to cow
insulin.
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 DRUG DEVELOPED FROM RECOMBINANT
DNA TECHNOLOGY COSTS HIGH AMOUNT
OF MONEY
o Interferon β-1b - treats a type of multiple
sclerosis (costs close to $70,000 per year)
o Tissue plasminogen activator (tPA) - a
recombinant clotbusting drug, also has
cheaper alternatives. If injected within 4
hours of a heart attack, tPA dramatically
limits damage to the heart muscle by
restoring blood flow. (t costs $2,500 a
shot)
 Safer vaccines are created using
recombinant DNA technology
o alternative flu vaccine (eggless vaccine)
consists of the genes that encode the
hemagglutinin proteins from two
influenza A strains and one influenza B
strain (effective against H1N1 and H3N2);
also does not use antibiotics,
preservatives, or live flu viruses, and can
be manufactured faster than
conventional flu vaccine.
o new vaccine that protects against malaria
is based on altering a bacterium (Pantoea
agglomerans) that normally inhabits the
mosquito gut. Mosquitoes transmit
Plasmodium falciparum, the parasite that
causes malaria.
 Using recombinant bacteria is easier
than genetically modifying
mosquitoes to prevent malaria
EPO: BUILT-IN BLOOD BOOSTER OR PERFORMANCE-
ENHANCING DRUG?
 A glycoprotein hormone that the kidneys
produce in response to low levels of oxygen
in the blood.
 Travels to the bone marrow and binds
receptors on cells that give rise to red blood
cell progenitors.
 Enhance stamina results
 Causes severe anemia
 Difficult to boost because levels in human
plasma are too low to pool from donors.
 Valued as a dug after the invention of
hemodialysis—Removes EPO from the
Blood.
 Can be obtain from urine that has abundant
EPO secreted by South American Farmers
with hookworm infections and Japanese
aplastic anemia patients.
 Using Recombinant DNA technology, EPO is
now able to treat anemia in dialysis and AIDS
patients which is given with cancer
chemotherapy to avoid the need for
transfusions.
 Thickens the blood
 Raising the risk of a blockage that can cause
a heart attack or stroke, especially when
intense, grueling exercise removes water
from the bloodstream.
 People with any of four types of familial
erythrocytosis get the effects of extra EPO
naturally
o may cause dizziness, headaches,
nosebleeds, and shortness of breath, or
have no symptoms, but it increases the
risk of stroke and heart attack from
blocked circulation
o Autosomal recessive form increases the
level of EPO in the bloodstream
Transgenic Organisms
 Body fluid from transgenic animals is a better
way to express some recombinant genes.
o The genetic change must be introduced
into a fertilized ovum so that it is present
in every cell of the transgenic organism.
 Transgenic sheep, cows, and goats have all
expressed human genes in their milk,
including genes that encode clotting factors,
clot busters, and the connective tissue
protein collagen.
 Production of human antibodies (used to
treat cancer) from rabbit and cow milk
illustrates the potential value of transgenic
animals by attaching the appropriate human
antibody genes to promoters for milk
proteins.
NOTE: Antibodies are assembled from the
products of several genes.
 Technique used to insert DNA into animal
cells to create transgenic animals: Chemicals
and brief jolts of electricity - open transient
holes in plasma membranes that admit
“naked” DNA, or a gun-like device is used to
shoot tiny metal particles coated with DNA
inside cells. DNA may also cross the plasma
membrane in tiny fatty bubbles called
liposomes.
 Steps in creating a transgenic animal:
1. Foreign DNA into a fertilized ovum
2. Recombinant DNA will enter the nucleus
3. Replicate with cell’s own DNA
4. Transmission when cell divides
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 5. For ANIMALS: regeneration through
gestation in a surrogate mother.
CONDITIONS:
a. Dominant Trait: the transgenic
organism must express it in the
appropriate tissues at the right time
in development to be useful.
b. Recessive trait: crosses between
heterozygotes may be necessary to
yield homozygotes that express the
trait.
6. The organism will pass the characteristic
on to the next generation.
Animal Models
 Drug candidates can be tested on these
animal models and abandoned before being
tested in humans if they cause significant
side effects.
 Inserting the mutant human beta globin gene
that causes sickle cell disease into mice
results in a mouse model of the disease.
 Limitations:
a. Researchers cannot control where a
transgene inserts in a genome, and how
many copies insert, so that different
animals may have different numbers of
the gene of interest.
b. The level of gene expression necessary
for a phenotype to emerge may also
differ in the model and humans.
c. Animal models might not mimic the
human condition exactly because of
differences in rates of development. (eg.,
some inherited diseases that do not
cause symptoms until adulthood in
humans, mice simply do not live long
enough to have the associated
phenotype.)
 a transgenic monkey is a more accurate
model of Huntington disease than a mouse,
because as a primate the monkey is much
more similar to a human in life span,
metabolism, reproduction, behavior, and
cognition
Genetically Modified Foods
 Seedless fruit & lean meat - a form of genetic
modification based on phenotype, such as
taste or appearance, and is therefore both
subjective and imprecise, affecting many
genes
 Genetically modified organisms (GMOs)-
Organisms altered to have genes from other
species or to over- or underexpress their own
genes
o Golden rice - a well-known GM crop that
uses genes from corn and bacteria to
produce 23 times as much betacarotene
(a vitamin A precursor) as unaltered rice.
 improve human nutrition in the
many nations where rice is a dietary
staple
 contains no allergens or toxins
 Developed by the not-for-profit
International Rice Research Institute
in the Philippines
 Cons of GM crops:
o Labeling a food that contains a nutrient
not normally in it—like a protein from
peanuts in corn—could prevent allergic
reactions
o field tests may not adequately predict the
effects on ecosystems.
o Some people fear GM foods.
o overreliance on them may lead to genetic
uniformity, which is just as dangerous as
traditional monoculture
 Many GM crops are designed to resist certain
herbicides (a pesticide used to kill unwanted
plants)
Bioremediation
 bacteria or plants with the ability to detoxify
certain pollutants are released or grown in a
particular area.
 Uses genes that enable an organism to
metabolize a substance that, to another
species, is a toxin.
 Uses unaltered organisms, and also transfers
“detox” genes to other species so that the
protein products can more easily penetrate a
polluted area.
 Transgenic microorganisms that make
proteins that detoxify contaminants are sent
into plants whose roots distribute the detox
proteins in the soil.
 Also used as cleaning up munitions dumps
from wars such as deploying bacteria that
normally break down trinitrotoluene, or TNT,
the major ingredient in dynamite and land
mines (The enzyme used is linked to the GFP
gene from jellyfish)
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 MONITORING GENE FUNCTION
 Gene expression is where we can make a
difference by controlling our environment.
 Monitoring gene expression requires
detecting the mRNAs in particular cells under
particular conditions.
 Gene expression DNA microarrays, or gene
chip- device that can detect and display the
mRNAs in a cell; chooses the types of cells to
interrogate
 DNA microarray - a piece of glass or plastic
that is about 1.5 centimeters square—
smaller than a postage stamp.
 COMPARISON OF AN INJURED PERSON
(CSF) FROM A HEALTHY PERON (CSF)
o Researchers make cDNAs from mRNA
using an enzyme from a retrovirus,
reverse transcriptase.
o The cDNAs from the injury sample are
labeled with a red fluorescent dye and the
cDNAs from the control sample are
labeled with a green fluorescent dye
which are then applied to the microarray.
o A computer algorithm interprets the
pattern of gene expression
o Visual data for spinal cord sample:
a. RED- a gene expressed in CSF only
when the spinal cord is injured (and
presumably leaking inflammatory
molecules).
b. GREEN- a gene expressed in CSF only
when the spinal cord is intact.
c. YELLOW- positions where both red-
and greenbound dyes fluoresce,
representing genes that are
expressed whether or not the spinal
cord has been injured.
d. BLACK/LACK OF FLUORESCENCE-
corresponds to DNA sequences that
are not expressed in CSF because they
do not show up in either sample.
GENE SILENCEING AND GENOME EDITING
 These biotechnologies are based on the
phenomenon of complementary base pairing
and some borrow directly from natural DNA
repair pathways.
GENE SLICING
 Block synthesis of, or degrade, mRNA.
 Antisense technology – a form of gene
silencing that blocks expression of a gene by
introducing RNA that is complementary to
the gene’s mRNA transcript
 Antisense RNA- introduced RNA; binds to the
mRNA, preventing its translation into
protein.
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 Morpholinos- second variation of antisense
technology uses synthetic molecule; consist
of sequences of 25 DNA bases attached to
organic groups that are similar to, but not
exactly the same as, the sugar-phosphate
backbone of DNA.
o Can block splice-site mutations that
would otherwise delete entire exons.
o treat Duchenne muscular dystrophy, but
is still being tested to see if it can restore
enough dystrophin to provide sustained
improvement in mobility.
 Ribozymes- another approach in gene
splicing; These are RNA molecules that are
part of ribosomes (the organelles on which
translation occurs) that have catalytic
activity, like enzymes.
o fit the shapes of certain RNA molecules.
o cut RNA, they can be used to destroy
RNAs from pathogens, such as HIV
RNA Interference (RNAi)
 RNA interference (RNAi)- a gene silencing
technique based on the fact that RNA
molecules can fold into short, double-
stranded regions where the base sequence is
complementary.
o Short, double-stranded RNAs sent into
cells by small interfering RNAs (siRNAs)
and separates into single strands, one of
which binds its complement in mRNA,
preventing it from being translated.
 Dicer- an enzyme that cuts long, double-
stranded RNAs into pieces 21 to 24
nucleotides long.
o The RNA pieces contact a group of
proteins that form an RNA-induced
silencing complex, or RISC.
 Guide strand, adheres- One strand of the
double-stranded short RNA
o guide strand finds its complementary RNA
and binds as part of RISC.
 Argonaute- a protein which is another part
of RISC is responsible for degrading the
targeted RNA, preventing its translation into
protein.
 SiRNA – have the capabilities to alter
methylation and the binding of histones to
certain genes.
o Has a synthetic type which carries out
RNAi and can inflame the liver, rather
than reach their intended targets.
NOTE:
(1) creating vaccines by knocking down
expression of key genes in viruses that cause
diseases
(2) treating cancer by silencing oncogenes
(3) creating a better-tasting decaf coffee by
silencing an enzyme required for caffeine
synthesis in coffee plants.
GENOME EDITING
 Uses restriction endonucleases to cut and
paste DNA molecules in patterns that might
not exist in nature.
 create double-stranded breaks in the DNA
double helix, enabling insertion of a desired
DNA sequence or removal of a sequence.
 the enzymes generate double-stranded
breaks, which natural DNA repair systems
mend, removing or even replacing or adding
to the targeted area
 can be done on somatic cells or on cells of the
germline
o Somatic applications: the change must
affect all or enough cells to alter the
phenotype, such as treating symptoms of
a genetic disease
o Germline genome editing: introduces the
genetic change to every cell in an
organism because it is carried out at the
beginning of development.
 THREE MAJOR GENOME EDITING
TECHNIQUES: [all are more precise than gene
therapies]
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 Endonuclease- severs both strands of the dengue fever, and yellow fever, and
double helix at the same point. the Anopheles species that transmit malaria.
Zinc Finger Nuclease [ZFNs] Technology  Gene drive- The application of genome
editing to kill, alter, or render infertile a
 Zinc Fingers- motif protein that is used;
pathogen
consist of a beta-pleated sheet linked to an
o based on a natural form of DNA repair,
alpha helix by a zinc atom
called homing (found in certain single-
o ACTION: a little like folding a small loop
celled organisms), which removes one of
of yarn and snipping it across both strands
a pair of alleles of a selected gene and
with one cut.
replaces it with another copy of the
 Different zinc fingers bind different three- remaining allele.
base DNA sequences o DNA sequence of 15 to 30- The proteins
 If zinc fingers bind, then another nuclease that they encode recognize, cut them out,
(FokI) cuts the DNA. and replace them with a copy of the DNA
 In: Transcription-activator-like effector sequence on the other strand.
nuclease (TALEN) technology- a restriction o homing proteins are incorporated into
enzyme from a bacterium (Xanthomonas) CRISPR-Cas9 to delete and then replace a
that infects plants cuts DNA on both strands selected gene.
 Chimeric antigen receptor technology- o An incredibly fast way to alter a wild
treats cancer; used ZFNs at first but switched population, research is restricted to
to CRISPR-Cas9 technology. highly controlled, laboratory settings.
CRISPR-Cas9 o May not be permanent after changes of
 use proteins and can only cut one gene at a the genetic structure of a population due
time. to evolution.
o uses RNA and is cheaper and easier to o Approach in mosquito populations: alter
use, and can remove, replace, or add the germline
more than one gene at a time.
 Short sequences of DNA that include several
repeats.
 CRISPRs are natural components of the
genomes of certain bacteria, where they
provide an action similar to an immune
response.
o enable bacteria to deploy a restriction
enzyme called Cas9 to recognize and cut
out DNA sequences from infecting viruses
that have inserted into the bacterial
genome
 CRISPR DNA sequences along with the viral
sequences are transcribed into CRISPR RNAs
and then serve as “guides” that bring Cas9 to
other sites where viral DNA has integrated
into the bacterial genome.
 CRISPR RNAs- remain to eject DNA from
future viral infections.
 Has great versatility— researchers can
design and synthesize guide RNAs to direct
which DNA sequences are removed, and
even stitch in desired sequences to replace
the ones snipped out, or add new genes.
 Works in ANY SPECIES
 Treating genetic disease and creating animal
models
 Top target: MOSQUITOES particularly Aedes
aegypti, which spreads Zika virus disease,

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