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Chapter 18
DNA Technologies:
Making and Using
Genetically Altered
Organisms, and
Other Applications
A Molecular Biology Laboratory
Why It Matters…
• Techniques used to isolate, purify, analyze, and
manipulate DNA sequences are known as DNA
technologies
• Scientists use DNA technologies both for basic
research into the biology of organisms and for
applied research
• The use of DNA technologies to alter genes for
practical purposes is called genetic engineering
Biotechnology
• Genetic engineering is part of biotechnology –
any technique applied to biological systems or
living organisms to make or modify products or
processes for a specific purpose
• Biotechnology also includes non-DNA technologies
such as the use of yeast to brew beer and the use
of bacteria to make yogurt and cheese
• Biotechnology today often includes genomics (the
characterization of whole genomes) and
bioinformatics tools (application of mathematics
and computer science to biological data)
18.1 Key DNA Technologies for Making
Genetically Altered Organisms
• DNA cloning is a method for producing many
copies of a piece of DNA
• When DNA cloning involves a gene, it is called
gene cloning
• One common method for cloning a gene of
interest is to insert it into plasmids, producing
recombinant DNA molecules
• The plasmids are inserted into bacteria, which
replicate the recombinant DNA as they grow and
divide
Genetically Modified Organisms
• Any organism that has its genome altered to
change a genetic trait or traits is a genetically
altered organism
• Genetically modified organisms (GMOs) have
their genomes specifically engineered to introduce
or change a genetically controlled trait
• GMOs contain recombinant DNA—DNA
fragments from two or more different sources that
have been joined together to form a single
molecule
Cloned DNA in Research
• Cloned DNA may be used in basic research to
study gene structure or function, including how its
expression is regulated, and the nature of the
gene’s product
• Cloned DNA may be used in applied research for
medical, forensic, agricultural, or commercial
applications:
• Gene therapy or diagnosis of genetic diseases
• Production of pharmaceuticals
• Production of genetically modified animals and
plants
• Modification of bacteria to clean up toxic waste
Restriction Enzymes
• Bacterial enzymes called restriction
endonucleases (restriction enzymes) are used
to join two DNA molecules from different sources
• Restriction enzymes recognize specific DNA
sequences (restriction sites) and cut the DNA at
specific locations within those sites
• The DNA fragments produced by a restriction
enzyme are known as restriction fragments
Restriction Enzymes (cont'd.)
• Restriction enzymes cut DNA at a specific
restriction site
• The sequence of nucleotides (read in the 5′→3′
direction) are the same on both strands
(symmetrical)
• The DNA fragments have single-stranded ends
(sticky ends) that hydrogen-bond with
complementary sticky ends on other DNA
molecules cut with the same enzyme
• The sugar–phosphate backbones of the DNA
strands are sealed by DNA ligase (ligation)
Bacterial Plasmids as Cloning Vectors
• Bacterial plasmids are examples of cloning
vectors – DNA molecules into which a DNA
fragment is inserted to form a recombinant DNA
molecule for the purpose of cloning
• Plasmid cloning vectors are engineered with two
genes used to locate bacteria that incorporate
recombinant plasmids:
• The ampR gene encodes an enzyme that breaks
down the antibiotic ampicillin
• The lacZ+ gene encodes β-galactosidase, which
hydrolyzes the sugar lactose
Cloning a Gene of Interest
• DNA fragments and plasmid, both cut within the
lacZ+ gene with the same restriction enzyme, are
mixed together with DNA ligase to produce
recombinant plasmids
• DNA molecules are transformed into ampicillinsensitive, lacZ– E. coli, which are spread on a
plate containing ampicillin and the β-galactosidase
synthetic substrate X-gal
• Bacteria that have been transformed with
recombinant plasmids are identified by blue-white
screening
Polymerase Chain Reaction (PCR)
• The polymerase chain reaction (PCR) produces
an extremely large number of copies of a specific
DNA sequence without having to clone the
sequence in a host organism
• PCR is essentially DNA replication in which a DNA
polymerase replicates only a portion of a DNA
molecule
• The primers used in PCR are designed to isolate
the sequence of interest – by cycling 20 to 30
times through a series of steps, PCR amplifies the
target sequence, producing millions of copies
Gel Electrophoresis
• Gel electrophoresis is a technique that separates
DNA, RNA, or protein molecules in a gel subjected
to an electric field – based on size, electrical
charge, or other properties
• PCR results can be compared using agarose gel
electrophoresis – the size of the amplified DNA is
determined by comparing the position of the DNA
band with the positions of bands of a DNA ladder
Review of Key Concepts in This Section
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Gene cloning
Recombinant DNA
Restriction enzyme (restriction endonuclease)
Ligation
DNA ligase
Cloning vector
Polymerase chain reaction (PCR)
Agarose gel electrophoresis
18.2 Applications of Genetically Altered
Organisms
• DNA technologies are used in research:
• Cloning genes to determine their structure,
function, and regulation of expression
• Manipulating genes to determine how their
products function in cellular or developmental
processes
• Identifying differences in DNA sequences among
individuals in ecological studies
• DNA technologies also have practical applications:
• Medical and forensic detection, modification of
animals and plants, and manufacture of
commercial products
Genetic Engineering
• Genetic engineering uses DNA technologies to
modify genes of a cell or organism – organisms
that receive genes from an external source
(transgenes) are called transgenic
• Genetic engineering has been used to produce
proteins used in medicine and research; to correct
hereditary disorders; and to improve animals and
crop plants
• Some people have ethical concerns, or fear that
the methods may produce toxic or damaging
foods, or release dangerous and uncontrollable
organisms
Engineering Bacteria to Produce Proteins
• Engineering E. coli to make a foreign protein:
• The protein-coding sequence of a gene is inserted
into an expression vector (plasmid) which
contains regulatory sequences that allow
transcription and translation of the gene
• The recombinant plasmid is transformed into E. coli
• The inserted gene is expressed in E. coli,
transcribed, and translated to make the encoded
eukaryotic protein
• The protein is extracted from bacterial cells and
purified, or purified from the culture medium
Cloning Eukaryotic Genes in Bacteria
• Most eukaryotic protein-coding genes have
introns, which are absent in bacterial and most
archaeal protein-coding genes
• After mRNA has been processed in eukaryotes, a
double-stranded DNA copy can be made by
reverse transcription
• The enzyme reverse transcriptase can make this
copy and is used in reverse transcriptase-PCR
• This complementary DNA (cDNA) copy can then
be cloned and expressed in bacterial cells
Genetic Engineering of Animals
• Gene targeting is the knocking out, replacement,
or addition of a gene in a genome
• Gene targeting methods have been developed for
a number of model animals
• Many methods require the use of stem cells to
produce the GMO
Stem Cells
• Stem cells are cells capable of undergoing many
divisions in an undifferentiated state, and also have
the ability to differentiate into specialized cell types
• Adult stem cells function to replace specialized
cells in various tissues and organs
• These cells are multipotent—they have a restricted
ability to produce only certain cell types
• Embryonic stem cells are found in an early-stage
embryo (blastocyst) and can differentiate into all of
the tissue types of the embryo
• These cells are pluripotent
Gene Targeting in Mice
• In mice, transgenes are introduced into embryonic
stem cells, which are then injected into early-stage
embryos
• The stem cells differentiate into a variety of tissues
along with embryonic cells, including sperm and
egg cells
• Males and females are bred, leading to offspring
that contain one or two copies of the introduced
gene
• A knockout mouse is a homozygous recessive
that receives two copies of a gene altered to a
nonfunctional state
Focus on Research: Programmable RNAGuided Genome Editing System
• CRISPR loci and cas genes together encode an
immune system against foreign bacteriophages
and plasmids in bacterial and archaeal cells
• The natural CRISPR-Cas system has been
modified to be a programmable RNA-guided
genome editing system for research purposes
• This technology has been embraced rapidly by
research groups for both basic and applied
research
• Example: making gene knockouts is simpler and
more time-efficient with CRISPR-Cas than
traditional methods
Gene Therapy
• Gene therapy is the introduction of a normal gene
into particular cell lines to correct genetic disorders
• Germline gene therapy is the experimental
introduction of a gene into germline cells of an
animal
• This type of therapy is not allowed in humans
• Humans are treated with somatic gene therapy
• Somatic cells are cultured and transformed with an
expression vector containing the transgene
• Modified cells are reintroduced into the body
Gene Therapy in Humans
• Somatic gene therapy has been successfully used
to treat specific cases of adenosine deaminase
deficiency (ADA)
• Other somatic gene therapy trials have ended
badly:
• A teenage patient died as a result of a severe
immune response to the viral vector being used
• Several children in gene therapy trials using
retrovirus vectors have developed a leukemia-like
condition
Turning Domestic Animals into
Protein Factories
• Genetic engineering can turn animals into
pharmaceutical factories for production of proteins
used to treat human diseases or other medical
conditions (e.g., clotting factor)
• Most animals are engineered to produce proteins
in milk, making purification easy, and harmless to
the animals
• Pharming projects are underway for proteins to
treat cystic fibrosis, collagen for wrinkles, human
milk proteins for infant formulas, and normal
hemoglobin for blood transfusions
Producing Animal Clones
• 1997: Ian Wilmut and Keith Campbell successfully
cloned a sheep (“Dolly”) using a somatic cell from
an adult sheep
• Several commercial enterprises now provide
cloned copies of champion animals
• Cloned animals often suffer from abnormal
conditions – genes may be lost or may be
expressed abnormally
• Molecular studies show that the expression
hundreds of genes in the genomes of clones may
be regulated abnormally
Genetic Engineering of Plants
• Plants are engineered for increased resistance to
pests and disease; greater tolerance to heat,
drought, and salinity; larger crop yields; faster
growth; and resistance to herbicides
• Individual adult cells of some plants can be altered
by the introduction of a desired gene, then grown
in cultures into a multicellular mass of cloned cells
called a callus
• The callus forms a transgenic plant with the
introduced gene in each cell
Methods Used to Insert Genes into Plants:
The Ti Plasmid
• A tumor of deciduous trees (crown gall disease) is
caused by the bacterium Agrobacterium
tumefaciens, which contains a large, circular
plasmid – the Ti (tumor-inducing) plasmid
• Genes on a segment of the Ti plasmid (T DNA)
integrate into the plant genome and are
expressed; the products stimulate cell growth and
division, producing a tumor
• The Ti plasmid is used as a vector for making
transgenic plants (similar to bacterial plasmids
used in bacteria)
Plant Genetic Engineering Projects
• Genetic engineering is used to produce transgenic
crops – at least two-thirds of the processed, plantbased foods sold at many national supermarket
chains contain transgenic plants
• Crops such as corn, cotton, and potatoes have
been modified for insect resistance by introduction
of the bacterial gene for Bt toxin, a natural
pesticide
• Papaya and squash have been genetically
engineered for virus resistance
Plant Engineering Projects (cont'd.)
• Several crops have been engineered for
resistance to herbicides – most corn, soybean,
and cotton plants grown in the US are glyphosateresistant (“Roundup-ready”) varieties
• Crop plants are also being engineered to alter
nutritional qualities – “golden rice” contains genes
for synthesis of β-carotene, a precursor of vitamin
A
• Plant pharming of transgenic plants to produce
medically valuable products is being developed
Molecular Insights: Nutritional Quality of
Genetically Modified Food
• Research Question: Are the metabolomes of
genetically modified foods significantly different
from their unmodified (transgenic) versions?
• Conclusion: Genetically modifying tomatoes to
delay fruit ripening does not significantly affect the
metabolic fingerprint of the tomato fruit other than
the fruit-ripening metabolites
Public Concerns About Genetic Engineering
• When recombinant DNA technology was
developed, one key concern was that a bacterium
carrying a recombinant DNA molecule might
escape into the environment, transfer the
recombinant molecule to other bacteria, and
produce new, potentially harmful, strains
• To address these concerns, U.S. scientists drew
up comprehensive safety guidelines for
recombinant DNA research in the United States
Public Concerns About Genetic Engineering
(cont'd.)
• Issues include the safety of consuming GMOcontaining foods, and possible adverse effects on
the environment:
• GMOs interbreeding with natural species
• Beneficial insect species such as monarch
butterflies feeding on plants with Bt toxins
Global Reactions
• Different countries have reacted to GMOs in
different ways:
• In the U.S., GMOs are evaluated for potential risk
by appropriate government regulatory agencies
• In the EU, all use of GMOs in the field or in food
requires authorization following a careful review
process
• On a global level, the Cartagena Protocol on
Biosafety “promotes biosafety by establishing
practical rules and procedures for the safe
transfer, handling and use of GMOs”
18.3 Other Applications of DNA Technologies
• Some applications for DNA technologies do not
involve making or using genetically altered
organisms
• Many genetic diseases are caused by defects in
enzymes or other proteins that result from
mutations at the DNA level
• Scientists can often use DNA technologies to
develop molecular tests for those diseases
• Example: The sickle-cell mutation changes a
restriction site in the DNA – cutting the β-globin
gene with MstII produces two DNA fragments
from the normal gene and one fragment from
the mutated gene
RFLPs
• Restriction enzyme-generated DNA fragments of
different lengths from the same region of the
genome are known as restriction fragment
length polymorphisms (RFLPs)
• RFLPs typically are analyzed using agarose gel
electrophoresis
• The single base-pair mutation in the b-globin gene
in sickle-cell anemia is an example of a singlenucleotide polymorphism (SNP)
• An SNP locus typically has two alleles; by definition
to be an SNP, the frequency of the rarer gene must
be at least 1%
DNA Fingerprinting
• Each human has unique combinations and
variations of DNA sequences known as DNA
fingerprints (except identical twins)
• DNA fingerprinting (also called DNA profiling) is
a technique used to distinguish between
individuals of the same species using DNA
samples
• DNA fingerprinting is commonly used for
distinguishing human individuals in forensics and
paternity testing
Principles of DNA Fingerprinting
• In DNA fingerprinting, PCR is used to analyze
DNA variations at various loci in the genome
• In the U.S., 13 loci in noncoding regions of the
genome are the standards for PCR analysis
• Each locus is an example of a short tandem
repeat (STR) sequence (or microsatellite) – a
short sequence of DNA repeated in series, with
each repeat 2-6 bp
Principles of DNA Fingerprinting (cont'd.)
• Each locus has a different repeated sequence,
and the number of repeats varies among
individuals in a population
• As a further source of variation, a given individual
is either homozygous or heterozygous for an STR
allele
• Because each individual has an essentially unique
combination of alleles, analysis of multiple STR
loci can discriminate between DNA of different
individuals
DNA Fingerprinting in Forensics
• DNA fingerprints are routinely used to identify
criminals or eliminate suspects in legal
proceedings
• DNA fingerprints might be prepared from hair,
blood, or semen found at the scene of a crime
• DNA fingerprinting of stored forensic samples has
led to the release of persons wrongly convicted of
rape or murder
• Typically, the evidence is presented in terms of
probability that a DNA sample came from a
random individual
DNA Fingerprinting in Paternity and Ancestry
• DNA fingerprints are widely used as evidence of
paternity because parents and their children share
common alleles
• DNA fingerprints are also used to confirm the
identity of human remains
• DNA fingerprints have been used to investigate
pathogenic E. coli in hamburger meat, in cases of
wildlife poaching, to detect genetically modified
organisms, and to compare the DNA of ancient
organisms with present-day descendants