Download Frontiers of Biotechnology

Frontiers of Biotechnology
PowerPoint Lectures for
Biology, Seventh Edition
Neil Campbell and Jane Reece
Lectures by Chris Romero
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Overview: Understanding and Manipulating Genomes
• Sequencing of the human genome was largely completed
by 2003
• DNA sequencing has depended on advances in
technology, starting with making recombinant DNA
– In recombinant DNA, nucleotide sequences from two
different sources, often two species, are combined in
vitro into the same DNA molecule
• Methods for making recombinant DNA are central to
genetic engineering, the direct manipulation of genes for
practical purposes
• In broad terms, biotechnology is the manipulation of
organisms or their components to make useful products
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
DNA cloning permits production of multiple copies of a
specific gene or other DNA segment
• To work directly with specific genes, scientists
prepare gene-sized pieces of DNA in identical
copies, a process called gene cloning
– Techniques for gene cloning are used to prepare
multiple identical copies of pieces of DNA.
– Most methods for cloning pieces of DNA in the
laboratory share general features, such as the use of
bacteria and their plasmids
• Cloned genes are useful for making copies of a
particular gene and producing a gene product
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Bacterium
Gene inserted into
plasmid
Bacterial
chromosome
Cell containing gene
of interest
Plasmid
Recombinant
DNA (plasmid)
Gene of
interest
Plasmid put into
bacterial cell
DNA of
chromosome
Recombinant
bacterium
Host cell grown in culture
to form a clone of cells
containing the “cloned”
gene of interest
Gene of
interest
Protein expressed
by gene of interest
Copies of gene
Basic
research
on gene
Gene for pest
resistance inserted
into plants
Protein harvested
Basic research and
various applications
Gene used to alter
bacteria for cleaning
up toxic waste
Protein dissolves
blood clots in heart
attack therapy
Basic
research
on protein
Human growth hormone treats stunted
growth
Using Restriction Enzymes to Make Recombinant DNA
• Bacterial restriction enzymes protect bacteria from the
DNA of phages or other bacteria by cutting up foreign DNA
in a process called restriction.
–
Restriction enzymes cut DNA molecules at DNA sequences called
restriction sites
–
A restriction enzyme usually makes many cuts, yielding restriction
fragments
• The most useful restriction enzymes cut DNA in a
staggered way, producing fragments with “sticky ends”
that bond with complementary “sticky ends” of other
fragments
• DNA ligase is an enzyme that seals the bonds between
restriction fragments
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
LE 20-2
Restriction site
DNA 5
3
3
5
Restriction enzyme cuts
the sugar-phosphate
backbones at each arrow.
Sticky end
DNA fragment from another
source is added. Base pairing
of sticky ends produces
various combinations.
Fragment from different
DNA molecule cut by the
same restriction enzyme
One possible combination
DNA ligase
seals the strands.
Recombinant DNA molecule
The Techniques of Gene Cloning
Cloning a human gene in a bacterial plasmid can
be divided into five steps:
1. Isolate a gene of interest, for example, the gene for human
insulin
2. Insert the gene into a bacterial plasmid
3. Insert the plasmid into a vector, a cell that will carry the
plasmid, such as a bacterium. To accomplish this, a bacterium
must be made competent (be able to take up the plasmid)
4. Clone the gene. As the bacteria reproduce themselves by
fission, the plasmid and the selected gene are also being
cloned. Millions of copies of the gene are being produced.
5. Identify the bacteria that contain the selected gene and harvest
it from the bacteria.
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
LE 20-3_3
Bacterial cell
Isolate plasmid DNA
and human DNA.
lacZ gene
(lactose
breakdown)
Human
cell
Restriction
site
ampR gene
(ampicillin
resistance)
Cut both DNA samples with
the same restriction enzyme.
Bacterial
plasmid
Gene of
interest
Sticky
ends
Human DNA
fragments
Mix the DNAs; they join by base pairing.
The products are recombinant plasmids
and many nonrecombinant plasmids.
Recombinant DNA plasmids
Introduce the DNA into bacterial cells
that have a mutation in their own lacZ
gene.
Recombinant
bacteria
Plate the bacteria on agar
containing ampicillin and X-gal.
Incubate until colonies grow.
Colony carrying nonrecombinant plasmid
with intact lacZ gene
Colony carrying
recombinant
plasmid with
disrupted lacZ gene
Bacterial
clone
Amplifying DNA in Vitro: The Polymerase Chain
Reaction (PCR)
• The polymerase chain reaction, PCR, can
produce many copies of a specific target segment
of DNA without using cells.
– A three-step cycle—heating, cooling, and
replication—brings about a chain reaction that
produces an exponentially growing population
of identical DNA molecules
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
LE 20-7
5
3
Target
sequence
Genomic DNA
Denaturation:
Heat briefly
to separate DNA
strands
Cycle 1
yields
2
molecules
Annealing:
Cool to allow
primers to form
hydrogen bonds
with ends of
target sequence
Extension:
DNA polymerase
adds nucleotides to
the 3 end of each
primer
Cycle 2
yields
4
molecules
Cycle 3
yields 8
molecules;
2 molecules
(in white boxes)
match target
sequence
3
5
5
3
3
5
Primers
New
nucleotides
Concept 20.2: Restriction Fragment Analysis
• Restriction fragment analysis detects differences in the
nucleotide sequences of DNA molecules
– Such analysis can rapidly provide comparative
information about DNA sequences
• In restriction fragment analysis, DNA fragments produced
by restriction enzyme digestion of a DNA molecule are
sorted by gel electrophoresis
– Restriction fragment analysis is useful for comparing
two different DNA molecules, such as two alleles for a
gene
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
LE 20-8
Cathode
Power
source
Mixture
of DNA
molecules
of different
sizes
Shorter
molecules
Gel
Glass
plates
Anode
Longer
molecules
Concept 20.3: Entire genomes can be mapped at
the DNA level
• The most ambitious mapping project to date has
been the sequencing of the human genome
– Officially begun as the Human Genome
Project in 1990, the sequencing was largely
completed by 2003
• Scientists have also sequenced genomes of other
organisms, providing insights of general biological
significance
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Concept 20.4: Genome sequences provide clues to
important biological questions
• In genomics, scientists study whole sets of genes
and their interactions
– Genomics is yielding new insights into genome
organization, regulation of gene expression,
growth and development, and evolution
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Identifying Protein-Coding Genes in DNA Sequences
• Computer analysis of genome sequences helps
identify sequences likely to encode proteins
• The human genome contains about 25,000 genes,
but the number of human proteins is much larger
• Comparison of sequences of “new” genes with
those of known genes in other species may help
identify new genes
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Concept 20.5: The practical applications of DNA
technology affect our lives in many ways
• Many fields benefit from DNA technology and genetic
engineering
– One benefit of DNA technology is identification of
human genes in which mutation plays a role in genetic
diseases
• Scientists can diagnose many human genetic disorders by
using PCR and primers corresponding to cloned disease
genes, then sequencing the amplified product to look for
the disease-causing mutation
• Even when a disease gene has not been cloned, presence
of an abnormal allele can be diagnosed if a closely linked
RFLP marker has been found
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Human Gene Therapy
• Gene therapy is the alteration of an afflicted
individual’s genes
– Gene therapy holds great potential for treating
disorders traceable to a single defective gene
• Vectors are used for delivery of genes into cells
• Gene therapy raises ethical questions, such as
whether human germ-line cells should be treated
to correct the defect in future generations
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
LE 20-16
Cloned gene
Insert RNA version of normal allele
into retrovirus.
Viral RNA
Retrovirus
capsid
Let retrovirus infect bone marrow cells
that have been removed from the
patient and cultured.
Viral DNA carrying the normal
allele inserts into chromosome.
Bone
marrow
cell from
patient
Inject engineered
cells into patient.
Bone
marrow
Forensic Evidence
• DNA “fingerprints” obtained by analysis of tissue
or body fluids can provide evidence in criminal and
paternity cases
– A DNA fingerprint is a specific pattern of bands
of RFLP markers on a gel
– The probability that two people who are not
identical twins have the same DNA fingerprint
is very small
– Exact probability depends on the number of
markers and their frequency in the population
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
LE 20-17
Defendant’s
blood (D)
Blood from defendant’s
clothes
Victim’s
blood (V)
Environmental Cleanup & Agricultural Productivity
• Genetic engineering can be used to modify the
metabolism of microorganisms
• Some modified microorganisms can be used to
extract minerals from the environment or degrade
potentially toxic waste materials
• DNA technology is being used to improve
agricultural productivity and food quality
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Animal Husbandry and “Pharm” Animals
• Transgenic organisms are made by introducing
genes from one species into the genome of
another organism
– Transgenic animals may be created to exploit
the attributes of new genes (such as genes for
faster growth or larger muscles)
– Other transgenic organisms are
pharmaceutical “factories,” producers of large
amounts of otherwise rare substances for
medical use
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Applications of Genetic Engineering
• Genetic engineering has spurred the growth of
biotechnology, a new industry that is changing the way we
interact with the living world.
– Transgenic bacteria now produce a host of important substances
useful for health & industry.
•
Human insulin, growth hormone, and clotting factor are now
produced by transgenic bacteria.
– Transgenic animals have been used to study genes and improve the
food supply.
•
Such animals often grow faster and produce less fatty meat.
– Transgenic plants are an important part of our food supply.
•
Many transgenic plants produce a natural insecticide, so the crops
do not have to be sprayed with pesticides.
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Cloning
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Safety and Ethical Questions Raised by DNA Technology
• Potential benefits of genetic engineering must be
weighed against potential hazards of creating
harmful products or procedures
• Most public concern about possible hazards
centers on genetically modified (GM) organisms
used as food
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings