Download Chapter 14 Biotechnology and Genomics

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Chapter 14
Biotechnology
and Genomics
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
DNA Cloning
• Cloning is the production of identical copies of
DNA through some asexual means.
– An underground stem or root sends up new shoots
that are clones of the parent plant.
– Members of a bacterial colony on a petri dish are
clones because they all came from division of the
same cell.
– Human identical twins are clones; the original single
embryo separate to become two individuals.
• Gene cloning is production of many identical
copies of the same gene.
– If the inserted gene is replicated and expressed, we
can recover the cloned gene or protein product.
– Cloned genes have many research purposes:
determining the base sequence between normal and
mutated genes, altering the phenotype, obtaining
the protein coded by a specific gene, etc.
– Humans can be treated with gene therapy:
alteration of the phenotype in a beneficial way.
– Otherwise transgenetic organisms are used to
produce products desired by humans.
– To clone DNA, scientists use recombinant DNA
(rDNA) and polymerase chain reaction (PCR).
Recombinant DNA Technology
• Recombinant DNA (rDNA) contains DNA
from two or more different sources.
• To make rDNA, technician selects a vector.
• A vector is a plasmid or a virus used to transfer
foreign genetic material into a cell.
• A plasmid is a small accessory ring of DNA in
the cytoplasm of some bacteria.
Fig. 14.1
• Plasmids were discovered in research on
reproduction of intestinal bacteria Escherichia
coli.
• The geneticists insert plasmids into antibioticsensitive bacterial cells by transformation.
• Introduction of foreign DNA into vector DNA
to produce rDNA requires two enzymes.
– Restriction enzyme is a bacterial enzyme that stops
viral reproduction by cleaving viral DNA.
•The restriction enzyme is used to cut DNA at specific
points during production of rDNA.
•It is called a restriction enzyme because it restricts growth
of viruses but it acts as a molecular scissors to cleave any
piece of DNA at a specific site.
•Restriction enzymes cleave vector (plasmid) and foreign
(human) DNA.
•Cleaving DNA makes DNA fragments ending in short
single-stranded segments with “sticky ends.”
•The “sticky ends” allow insertion of foreign DNA into
vector DNA.
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– DNA ligase seals the foreign gene into the vector
DNA
•When DNA splicing is complete, a rDNA (recombinant
DNA) molecule is formed.
•Treated cells take up plasmids, and then bacteria and
plasmids reproduce.
•Eventually, there are many copies of the plasmid and
many copies of the foreign gene.
• If the human gene is to express itself in a
bacterium, the gene must be accompanied by
the regulatory regions unique to bacteria and
meet other requirements.
– The gene cannot contain introns because bacteria
do not have introns.
– An enzyme called reverse transcriptase can be used
to make a DNA copy of mRNA.
– This DNA molecule is called complementary DNA
(cDNA) and does not contain introns.
– A laboratory DNA synthesizer can produce small
pieces of DNA without introns.
The Polymerase Chain Reaction
• PCR can create millions of copies of a single gene or a
specific piece of DNA in a test tube.
• PCR is very specific—the targeted DNA sequence can
be less than one part in a million of the total DNA
sample; therefore a single gene can be amplified using
PCR.
• PCR uses the enzyme DNA polymerase to carry out
multiple replications (a chain reaction) of target DNA.
• PCR automation is possible because heat-resistant
DNA polymerase from Thermus aquaticus, which
grows in hot springs, is an enzyme that withstands the
temperature necessary to separate double-stranded
DNA.
Fig. 14.2
• The PCR technique has virtually limitless applications.
It enables researchers to amplify and analyze tiny
DNA samples from a variety of sources, ranging from
crime scenes to archaeological remains.
DNA Analysis
• Gel electrophoresis is used for separating
macromolecules
– Mixtures of certain macromolecules—proteins,
polypeptides, DNA fragments, or RNA—are
separated by gel electrophoresis, a method that
exploits the fact that these molecules carry charged
groups that cause them to migrate in an electrical
field.
– Nucleic acids migrate through the gel toward the
positive pole of the electric field because they are
negatively charged due to their phosphate groups.
– The gel is stained after the DNA fragments are
separated to show their number and location.
• Restriction fragment length polymorphisms are
a measure of genetic relationships
– The variability of genes within a population can be
studied in several different ways. A traditional
approach is based on the fact that random DNA
mutations and recombination may result in
individuals with different lengths of fragments
produced by a given restriction enzyme. Such
restriction fragment length polymorphisms,
commonly known as RFLPs, can be used to
determine how closely related different members of
a population are.
– Restriction enzymes are used to cut the DNA from
two or more individuals, and the fragments are
separated by gel electrophoresis, with the DNA
from each individual in a separate lane; shorter
fragments migrate farther than longer fragments.
– RFLP analysis has been used when conducting
paternity testing and when analyzing evidence at
crime scenes. RFLP analysis has also been used to
help map the exact location of gene mutations.
– RFLP technique is rapidly being replaced by newer
methods, such as automated DNA sequencing.
One way to characterize DNA is to
determine its sequence of nucleotides
• Methods to rapidly sequence DNA exist
– Automated DNA sequencing relies on the chain
termination method.
– This method of DNA sequencing is based on the
fact that a replicating DNA strand that has
incorporated a modified synthetic nucleotide,
known as a dideoxynucleotide, cannot elongate
beyond that point. Unlike a “normal”
deoxynucleotide, which lacks a hydroxyl group on
its 2´ carbon, a dideoxynucleotide also lacks a
hydroxyl group on its 3´ carbon.
• The researcher prepares four different reaction
mixtures. Each contains multiple singlestranded copies of the DNA to be sequenced,
DNA polymerase, appropriate radioactively
labeled primers, and all four deoxynucleotides
needed to synthesize DNA: dATP, dCTP,
dGTP, and dTTP. Each mixture also includes
a small amount of only one of the four
dideoxynucleotides: ddATP, ddCTP, ddGTP,
or ddTTP.
• A mixture of DNA fragments of varying lengths
forms in the reaction mixture. The radioactive
fragments from each reaction are denatured
and then separated by gel electrophoresis, with
each reaction mixture, corresponding to A, T,
G, or C, occupying its own lane in the gel. The
positions of the newly synthesized fragments in
the gel can then be determined by
autoradiography.
• The chain termination method is still used in
automated
DNA-sequencing
machines.
However, the method of detection has changed
in that the nucleotide sequence is no longer
visualized by radiography. Instead, researchers
label each of the four ddNTPs with differentcolored fluorescent dye. The computer uses a
laser to read the fluorescence of the dye
markers as the bases emerge from the end of a
lane of electrophoresis medium.
• Entire genomes have been sequenced using automated
DNA sequencing
– Sequencing machines today are very powerful and can
decode about 1.5 million bases in a 24-hour period.
– These advances in sequencing technology have made it
possible for researchers to study the nucleotide sequences of
entire genomes in a wide variety of organisms, both
prokaryotic and eukaryotic.
– Human Genome Project
– By searching for DNA or amino acid sequences in a
database, researchers can gain a great deal of insight into the
function and structure of the gene product, the evolutionary
relationships among genes, and the variability among gene
sequences within a population.
Analyzing DNA
• Short tandem repeat (STR) profiling is a technique
used to analyze DNA.
• STRs are molecular markers that are short sequences
of repetitive DNA .
• Since the chromosomal location of STRs are known,
these are the only locations that are subjected to PCR
and gel electrophoresis.
• The band patterns are different in each person
because each individual has their own number of STR
repeats at different locations.
• The FBI uses a set of STRs from 13 different markers
to establish a unique DNA profile for an individual.
• DNA fingerprinting is a technique of using DNA
fragment lengths, resulting from restriction enzyme
cleavage and amplified by PCR, to identify particular
individuals.
• DNA is treated with restriction enzymes to cut it into
different sized fragments.
• During gel electrophoresis, fragments separate
according to length, resulting in a pattern of bands.
• DNA fingerprinting can identify deceased individuals
from skeletal remains, perpetrators of crimes from
blood or semen samples, and genetic makeup of longdead individuals or extinct organisms.
Fig. 14.3
• PCR amplification and DNA analysis is used to:
– detect viral infections, genetic disorders, and cancer;
– determine the nucleotide sequence of human
genes, and, because it is inherited, associate samples
with DNA of parents.
Biotechnology Products
• Genetically engineered organisms can produce
biotechnology products.
• Organisms that have had a foreign gene inserted
into them are transgenic.
Genetically Modified Bacteria
• Bacteria are grown in large vats called bioreactors.
– Foreign genes are inserted and the product is harvested.
– Products on the market include insulin, hepatitis B vaccine,
t-PA, and human growth hormone.
• Transgenic bacteria have been produced to protect
and improve the health of plants.
– Frost-minus bacteria protect the vegetative parts of plants
from frost damage.
– Root-colonizing bacteria receive genes from bacteria for
insect toxin, protecting the roots.
– Bacteria that colonize corn roots can be endowed with genes
for insect toxin.
• Transgenic bacteria can degrade substances.
– Bacteria selected for ability to degrade oil can be
improved by bioengineering.
– Bacteria can be bio-filters to prevent airborne
chemical pollutants from being vented into the air.
– Bacteria can also remove sulfur from coal before it
is burned and help clean up toxic dumps.
– Bacteria can also be given “suicide genes” that
caused them to die after they have done their job.
• Transgenic bacteria can produce chemical products.
– Genes coding for enzymes can be manipulated to catalyze
synthesis of valuable chemicals.
– Phenylalanine used in artificial sweetener can be grown by
engineered bacteria.
• Transgenic bacteria process minerals.
– Many major mining companies already use bacteria to
obtain various metals.
– Genetically engineered “bio-leaching” bacteria extract
copper, uranium, and gold from low-grade ore.
Genetically Modified Plants
• Plant cells that have had the cell wall removed are
called protoplasts.
• Electric current makes tiny holes in the plasma
membrane through which genetic material enters.
• The protoplasts then develop into mature plants.
• Foreign genes now give cotton, corn, and potato strains
the ability to produce an insect toxin and soybeans are
now resistant to a common herbicide.
• Plants are being engineered to produce human
proteins including hormones, clotting factors, and
antibodies in their seeds; antibodies made by corn,
deliver radioisotopes to tumor cells and a soybean
engineered antibody can treat genital herpes.
Corn has been genetically modified to contain theBt gene, a
bacterial gene that codes for a protein with insecticidal properties;
Bt stands for the bacterium’s scientific name, Bacillus
thuringiensis. Bt corn does not need periodic sprays of chemical
insecticides to control the European corn borer.
• The United States is the world’s top producer
of transgenic crops, also known as genetically
modified (GM) crops. Currently, 80% of the
U.S. soybean crop, 70% of its cotton, and 38%
of its corn are GM crops.
• DNA technology also has the potential to
develop crops that are more nutritious. For
example, in the 1990s geneticists engineered
rice to produce high quantities of beta-carotene,
which the human body uses to make vitamin A.
Genetically Modified Animals
• Animal use requires methods to insert genes
into eggs of animals.
– It is possible to microinject foreign genes into eggs
by hand.
– Vortex mixing places eggs in an agitator with DNA
and silicon-carbide needles that make tiny holes
through which the DNA can enter.
– Using this technique, many types of animal eggs
have been injected with bovine growth hormone
(bGH) to produce larger fishes, cows, pigs, rabbits,
and sheep.
• Gene pharming (pharmaceuticals + farming) is
the use of transgenic farm animals to produce
pharmaceuticals; the product is obtainable from
the milk of females.
– Genes for therapeutic proteins are inserted into
animal’s DNA; animal’s milk produces proteins.
– Drugs obtained through gene pharming are planned
for the treatment of cystic fibrosis, cancer, blood
diseases, and other disorders.
Cloning Transgenic Animals
• For many years, it was believed that adult vertebrate
animals could not be cloned; the cloning of Dolly in
1997 demonstrated this can be done.
• Cloning of an adult vertebrate would require that all
genes of an adult cell be turned on again.
• Cloning of mammals involves injecting a 2n nucleus
adult cell into an enucleated egg.
• The cloned eggs begin development in vitro and are
then returned to host mothers until the clones are
born.
• Animal cloning is a difficult process with a low success
rate. The vast majority of cloning attempts are
unsuccessful, resulting in the early death of the clone.
Applications of Transgenic Animals
• Scientists are using transgenic animals to
illustrate that maleness is due to a section of
DNA called SRY (the sex determining region of
the Y chromosome).
• Transgenic animals are also being used to
investigate various treatments for diseases by
eliminating genes from these animals.
• Organ transplants from transgenic animal
donors to human recipients is called
xenotransplantation.
Fig. 14.6
Gene Therapy
• Doctors perform genetic tests to determine whether an
individual has a particular genetic mutation associated
with disorders.
• Gene therapy involves procedures to give patients
healthy genes to make up for a faulty gene.
• Gene therapy also includes the use of genes to treat
genetic disorders and various human illnesses.
• There are ex vivo (outside body) and in vivo (inside
body) methods of gene therapy.
Fig. 14.7
Ex Vivo Gene Therapy
• Children with severe combined immunodeficiency
(SCID) underwent ex vivo gene therapy.
– Lacking the enzyme ADA involved in maturation of T and
B cells, they faced life-threatening infections.
– Bone marrow stem cells are removed, infected with a
retrovirus that carries a normal gene for the enzyme ADA,
and returned.
– Use of bone marrow stem cells allows them to divide and
produce more cells with the same genes.
– Patients who undergo this procedure show significant
improvement.
• Gene therapy include treatment of familial
hypercholesterolemia where liver cells lack a
receptor for removing cholesterol from blood.
– High levels of blood cholesterol make the patient
subject to fatal heart attacks when young.
– A small portion of the liver is surgically removed
and infected with retrovirus with normal gene for
receptor.
– This has lowered cholesterol levels following the
procedure.
In Vivo Gene Therapy
• Cystic fibrosis patients lack a gene for trans-membrane
chloride ion carriers; patients die from respiratory tract
infections.
– Liposomes, microscopic vesicles that form when
lipoproteins are in solution, are coated with healthy cystic
fibrosis genes and sprayed into a patient’s nostrils.
– Various methods of delivery are being tested for
effectiveness.
• A gene for vascular endothelial growth factor (VEGF)
can be injected alone or within a virus into the heart to
stimulate branching of coronary blood vessels.
• Another strategy is to make cancer cells more
vulnerable, and normal cells more resistant, to
chemotherapy.
• Injecting a retrovirus containing a normal p53
gene–that promotes apoptosis–into tumors may
stop the growth of tumors.
DNA technology has raised safety concerns
• The possibility that an organism with undesirable
environmental effects might be accidentally produced
was of great concern.
• Scientists feared that new strains of bacteria or other
organism, with which the world has no previous
experience, might be difficult to control.
• A great deal of research now focuses on determining
the effects of introducing transgenic organisms into a
natural environment. Carefully conducted tests have
shown that transgenic organisms are not dangerous to
the environment simply because they are transgenic.
• Environmental concerns about transgenic
animals also exist. Several countries are in the
process of developing fast-growing transgenic
fish, usually by inserting a gene that codes for a
growth hormone. The transgenic fish do not
grow larger than other fish, just faster. The
benefits of such genetically enhanced fish
include reduced pressure on wild fisheries and
less pollution from fish farms.
Genomics
• Genetics in the 21st century concerns genomics: the
study of genomes of humans and other organisms.
• Genomics is the emerging field of biology that studies
the entire DNA sequence of an organism’s genome to
identify all the genes, determine their RNA or protein
products, and ascertain how the genes are regulated.
• There are approximately 6 billion base pairs in the 2n
human genome.
• Genomics has three main areas of interest: structural
genomics, functional genomics, and comparative
genomics.
• Structural genomics is concerned with mapping
and sequencing genomes.
• Functional genomics deals with the functions of
genes and non-gene sequences in genomes.
• Comparative genomics involves comparing the
genomes of different species to determine what
changes in DNA have occurred during the
course of evolution.
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Sequencing the Genome
•
•
•
•
The Human Genome Project has produced a record
of all the base pairs in all our chromosomes.
The task took 13 years to learn the sequence of the
three billion base pairs along the length of our
chromosomes.
Determining that humans have 20,500 genes
required a number of techniques, many of which
relied on identifying RNAs in cells and then working
backward to find the DNA that can pair with that
RNA.
Following functional genomics, structural genomics
investigates base sequences and the number of genes
in humans.
Genome Comparisons
•
•
•
•
There is little difference between the sequence of
our bases and other organisms whose DNA
sequences are known.
We share a large number of genes with simpler
organisms (e.g., bacteria, yeast, mice); perhaps our
uniqueness is due to regulation of these genes.
Researchers found that certain genes on
chromosome 22 differed in humans and
chimpanzees: those for speech development,
hearing, and smell.
Many genes found were responsible for human
diseases.
• Genome analysis of commercially important
organisms, such as salmon, chickens, pigs, and
rice, is also a priority.
Eukaryotic Gene Structure
•
•
•
Eukaryotic chromosome structure is much more
complex than prokaryotic chromosome structure.
Generally speaking, more complex organisms have
more complex genes with more and larger introns.
The presence of introns allows exons to be put
together in various sequences so that different
mRNAs and proteins can result from a single gene.
Introns are currently regarded as gene expression
regulators and determine which genes are expressed
and how they are spliced.
Intergenic Sequences
•
•
•
•
Intergenic sequences are DNA sequences that occur
between genes.
Generally speaking, as the complexity of an
organism increases, so does the proportion of its
non-protein-coding DNA sequences.
Most of the human chromosomes are comprised of
intergeneic sequences, genes represent 1.5% of
human’s total DNA, and the remainder represents
“junk DNA,” which scientists believe serve many
important functions.
Three types of intergenic sequences found in the
human genome are: repetitive elements, tranposons,
and unknown sequences.
Fig. 14.8
Repetitive Elements
• Repetitive DNA elements occur when the same
sequence of two or more nucleotides are repeated
many times along the length of one or more
chromosomes.
• They make up nearly half of the human genome and
scientists believe that their true significance have yet to
be discovered.
• Repetitive DNA elements occur as tandem repeats –
meaning the repeated sequences are next to each other
on the chromosome.
• One type of tandem repeat sequence, short tandem
repeats (STRs), are a standard method in forensic
science for identifying one individual from another
and also determining family relationships.
• Another type of repetitive DNA element is called an
interspersed repeat – meaning the repetitions may be
placed intermittently along a single chromosome, or
across multiple chromosomes.
• Interspersed repeat are common and thought to play a
role in the evolution of new genes.
Transposons
• Transposons are specific DNA sequences that
move with and between chromosomes.
–
Their movement may increase or decrease the
expression of neighboring genes.
Unknown Sequences
• Unknown DNA sequences were once
dismissed as junk DNA since scientists could
not identify any function they have.
• Recently scientists observed that many of the
unknown sequences is transcribed into RNA.
• This RNA may carry out regulatory functions
more easily than proteins at times.
What Is a Gene?
• A modern definition of a gene, suggested by Mark
Gerstein and associates, “…is a genomic sequence
(either DNA or RNA) directly encoding functional
products, either RNA or protein.”
• This definition recognizes that the genetic material
involved does not have to be DNA, and that coding
does not only mean DNA sequencing, but the gene
product could be RNA or protein as well.
Proteomics
•
•
•
•
Proteomics is the study of the structure, function,
and interaction of cellular proteins.
Computer modeling of the 3-D shape of cellular
proteins is also an important part of proteomics.
Every somatic cell in the human body has essentially
the same genome, but cells in different tissues vary
greatly in the kinds of proteins they produce. Protein
expression patterns vary not only in different tissues
but also at different stages in the development of a
single cell.
The information obtained from proteomic studies
can be used in designing better drugs, and to
correlate drug treatment to the particular genome of
the individual.
Bioinformatics
• Bioinformatics is the application of computer
technologies to the study of the genome. It
includes the storage, retrieval, and comparison
of DNA sequences within a given species and
among different species.
• Information obtained from computer analysis
of the genome can show relationships between
genetic profiles and genetic disorders.